Antibodies Against Tissue Factor Pathway Inhibitor

ABSTRACT

The invention relates to antibodies that specifically bind to tissue factor pathway inhibitor (TFPI) and that reduce the clotting time of blood. Such antibodies have utility in the treatment of subjects with a coagulopathy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 13/140,296, filedAug. 30, 2011, which is a 35 U.S.C. §371 national stage application ofInternational Patent Application PCT/EP2009/067598 (published as WO2010/072691 A1), filed Dec. 18, 2009, which claimed priority of EuropeanPatent Application 08172520.2, filed Dec. 22, 2008; this applicationfurther claims priority under 35 U.S.C §119 of U.S. ProvisionalApplication 61/203,479, filed Dec. 23, 2008; the contents of allabove-named applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to antibodies that specifically bind totissue factor pathway inhibitor (TFPI).

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

In accordance with 37 C.F.R. §1.52(e)(5), Applicants enclose herewiththe Sequence Listing for the above-captioned application entitled“SEQUENCE LISTING”, created on May 26, 2011. The Sequence Listing ismade up of 46,035 bytes, and the information contained in the attached“SEQUENCE LISTING” is identical to the information in the specificationas originally filed. No new matter is added.—

BACKGROUND OF THE INVENTION

In subjects with a coagulopathy, such as in human beings withhaemophilia A and B, various steps of the coagulation cascade arerendered dysfunctional due to, for example, the absence or insufficientpresence of a coagulation factor. Such dysfunction of one part of thecoagulation cascade results in insufficient blood coagulation andpotentially life-threatening bleeding, or damage to internal organs,such as the joints. Subjects such as human beings with haemophilia A andB may receive coagulation factor replacement therapy such as exogenousFVIIIa or FIXa, respectively. However, such patients are at risk ofdeveloping “inhibitors” (antibodies) to such exogenous factors,rendering formerly efficient therapy ineffective. Furthermore, exogenouscoagulation factors may only be administered intravenously, which is ofconsiderable inconvenience and discomfort to patients. For example,infants and toddlers may have to have intravenous catheters surgicallyinserted into a chest vein, in order for venous access to be guaranteed.This leaves them at great risk of developing bacterial infections.Subjects with a coagulopathy may only receive therapy after a bleed hascommenced, rather than as a precautionary measure, which often impingesupon their general quality of life.

There are thus still many unmet medical needs in the haemophiliacommunity, in particular, and in subjects with coagulopathies, ingeneral.

When a vessel wall is injured, tissue factor (TF) is exposed to thecontents of circulating blood and TF forms a complex with FactorVII/activated Factor VII (FVII/FVIIa) on the surface of TF-bearingcells. This leads to the activation of Factor X (FX) to FXa whichtogether with FVa generates a limited amount of thrombin (FIIa). Smallamounts of thrombin activate platelets, which results in surfaceexposure of phospholipids that supports the binding of the tenasecomplex consisting of FVIIIa/FIXa.

The tenase complex produces large amounts of FXa, which subsequentlyfacilitates a full thrombin burst. A full thrombin burst is needed forthe formation of a mechanically strong fibrin structure andstabilization of the haemostatic plug. FVIII or FIX is missing orpresent at low levels in haemophilia patients, and due to the lack oftenase activity, the capacity to generate FXa is low and insufficient tosupport the propagation phase of the coagulation. In contrast, theTF-mediated initiation phase is not dependent on the formation of thetenase complex. However, the TF-pathway will, shortly after an initialFXa generation, be blocked by plasma inhibitors.

Tissue factor pathway inhibitor (TFPI) down-regulates ongoingcoagulation by neutralizing the catalytic activity of FXa and byinhibiting the TF-FVIIa complex in the presence of FXa. TFPI eitherinhibits the TF/FVIIa/FXa complex on the cellular surface or inhibitsreleased FXa followed by FVIIa/TF inhibition.

SUMMARY OF THE INVENTION

The Inventors have identified monoclonal antibodies which specificallybind to tissue factor pathway inhibitor (“TFPI”, sometimes referred toas “TFPI1”) and thereby modulate its activity. The present inventionrelates to these antibodies and to other related antibodies that arederived from these antibodies or have similar binding properties tothese antibodies.

Accordingly, the present invention relates to antibodies thatspecifically bind to tissue factor pathway inhibitor (TFPI) and thatreduce clotting time in, for example, (a) human FVIII-deficient plasmaand/or (b) human whole blood.

One antibody comprises the light chain variable region of SEQ ID NO: 4and the heavy chain variable region of SEQ ID NO: 8. Another antibodycomprises the light chain variable region of SEQ ID NO: 15 and the heavychain variable region of SEQ ID NO: 18.

The invention also provides polynucleotides which encode an antibody ofthe invention, such as polynucleotides which encode an antibody lightchain and/or an antibody heavy chain of the invention.

The invention also provides pharmaceutical compositions comprising anantibody or polynucleotide of the invention and a pharmaceuticallyacceptable carrier or diluent.

The antibodies, polynucleotides and compositions of the invention arealso provided for use in (a) the treatment or prevention of acoagulopathy (bleeding disorder) or (b) the stimulation of bloodclotting. That is, the invention provides a method for (a) the treatmentor prevention of a coagulopathy (bleeding disorder) or (b) thestimulation of blood clotting, the method comprising administering to apatient in need thereof a therapeutically or prophylactically effectiveamount of an antibody, polynucleotide or composition of the invention.

Furthermore, the invention provides dosing regimens of said monoclonalantibody of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the sequences of VH (A) and VL (B) domains of mouseanti-TFPI4F36A1B2 (herein also referred to as MuTFPI4F36 or 4F36),aligned with the sequences for the human germline and the initial CDRgrafted version of humanized TFPI4F36. The Kabat numbering scheme isindicated above the sequences.

FIG. 2 shows the nucleotide sequences and translated polypeptidesequences for the VH and VL sequences of the murine antibodyTFPI4F36A1B2 (MuTFPI4F36).

FIG. 3 shows the amino acid sequences of the light (A) and heavy (B)chains of Fab fragments of the murine 4F36 antibody, MuTFPI4F36.Numbering above the sequences is shown according to Kabat. Positionscorresponding to CDR loops are highlighted in bold underlined text inthe Kabat numbering Amino acid residues constituting the paratope arehighlighted in bold underlined text. The paratope is determined from theX-ray structure of the complex between the MuTFPI4F36 Fab and the TFPIK2 domain and is defined as residues in the Fab having a heavy atomwithin a distance of less than 4 A from a heavy atom in K2.

FIG. 4 shows the sequence of TFPI (signal peptide sequence omitted). TheKunitz domains are shown in bold: TFPI Kunitz domain 1=amino acids 26 to76; TFPI Kunitz domain 2=amino acids 97-147; TFPI Kunitz domain 3=aminoacids 188-238. The C-terminal part of TFPI is shown in italics at aminoacids 240 to 276.

FIG. 5 shows the relative accessibility of residues in TFPI. Theresidues that have a greater than 40% accessibility are amino acids94-95, 98, 100-110, 118-121, 123-124, 131, 134, 138-142 and 144-145.

FIG. 6 shows an SEC HPLC analysis of a complex between the TFPI Kunitzdomain 2 (K2) and the MuTFPI4F36 Fab fragment (Fab). SEC-HPLCchromatograms detected at UV 280 nm of free K2 (solid line, r_(t) 13.1min, peak shown at 13.134), free Fab (dashed line, r_(t) 11.7 min, peakshown at 11.676) and complex (dotted line, r_(t) 11.5 min, peak shown at11.496). The sample of the complex contained ˜20% excess K2.

FIG. 7 shows the overall structure of the MuTFPI4F36 Fab:K2 complex.Light chains are shown in pale grey and heavy chains are shown in darkgrey. The CDR loops as defined according to the Kabat scheme are labeledas L1 to L3 and H1 to H3.

FIG. 8 shows the structure of the K2 domain of TFPI when in a complexwith MuTFPI4F36 Fab (Fab molecule not shown). The N- and C-termini andsecondary structural elements are labeled.

FIG. 9 shows a back-bone superposition of K2 structures. Showsdifferences in structure between K2 solution, K2 is in complex withMuTFPI4F36 Fab and K2 in complex with porcine trypsin.

FIG. 10 shows the MuTFPI4F36 binding epitope on K2. (A) Cartoonrepresentation of the K2 domain of TFPI with side chains of residuesincluded in the binding epitope represented by balls and sticks. (B) isas A, but with surface added. (C) Binding epitope mapped on to primarysequence. Capital bold, italic and underlined letters corresponds toresidues in the K2-binding epitope making contacts with the MuTFPI4F36Fab heavy chain only (positions 10, 11, 13, 28, 31, 33 and 35), lightchain only (positions 21, 23 and 50), and with both heavy and lightchain (17, 19, 34 and 36), respectively. Secondary structural elements(h=helix, s=sheet) are indicated (helices at positions 5-8 and 50-56 andsheets at positions 20-26 and 31-37). Residues highlighted grey(positions 1-2 and 59-66) are present in the expressed protein, but arenot observed in the crystal structure due to the N- and C-termini beingflexible.

FIG. 11 shows a comparison of the back-bone traces of K2:MuTFPI4F36 Faband K2:HzTFPI4F36 Fab complexes, demonstrating the identical bindingmodes for the murine MuTFPI4F36 and humanized HzTFPI4F36 Fab fragments.K2:MuTFPI4F36 Fab is shown in grey and K2:HzTFPI4F36 Fab in black.Structures are superpositioned to optimize the match betwen the variableregion of the Fab fragments.

FIG. 12 shows the effect of anti-TFPI monoclonal antibodies (mAbs) onTF/FVIIa-induced activation of FX on the surface of HUVECs stimulatedwith TNFα/IL1β. Activation of FX was measured in the presence of 0-20 nMmAB (mAbTFPI 2021 or mAb 2974), 50 pM FVIIa (NovoSeven®) and 50 nM FX inbuffer with 25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, 5 mM CaCl₂, 1 mg/mlBSA (0.1%) pH 7.4 which was overlaid a monolayer of HUVECs. GeneratedFXa activity was determined in an amidolytic assay with S-2765 measuredby the increase in absorbance at 405 nM.

FIG. 13 shows the effect of anti-TFPI mAbs on TFPI inhibition ofTF/FVIIa-induced activation of FX on the surface of MDA-MB 231 cells.Activation of FX was measured in the presence of 0-20 nM mAb (Hz mAbTFPI2021 or mAb 2974), 2.5 nM fl-TFPI, 100 μM FVIIa and 50 nM FX in bufferwith 25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, 5 mM CaCl, 1 mg/ml BSA (0.1%)pH 7.4 which was overlaid a monolayer of MDA-MB 231 cells. Generated FXaactivity was determined in an amidolytic assay with S-2765 measured bythe increase in absorbance at 405 nM.

FIG. 14 shows the effect of single amino acid alanine substitutions ofselect residues within the TFPI Kunitz 2 domain on binding to mAbTFPI2021 (“mAb4F36”) and mAb2974 (n=2). The selected residues are part ofthe mABTFPI 2021 binding epitope. The numbering of amino acid residuesis as indicated in FIG. 10C.

FIG. 15 shows the cuticle bleeding time and blood loss measured intransient haemophiliac rabbits following treatment with control IgG(Haemophilia) or with the murine anti-TFPI antibody, TFPI-4F36A1B2(“4F36”, MuTFPI4F36).

FIG. 16 shows the cuticle bleeding time (single observations; mean±SEM)and blood loss (mean+SEM) in an “on demand” treatment of rabbits withantibody-induced haemophilia, treated with HzTFPI4F36 (“anti-TFPI”,mAbTFPI 2021) (2 mg/kg) or NovoSeven (9 mg/kg) 5 minutes after inductionof bleeding. The bleeding was observed for 1 hour (3600 sec).

FIG. 17 shows the cuticle bleeding time (single observations; mean±SEM)and blood loss (mean±SEM) in rabbits with antibody-induced haemophilia,when pre-treated with HzTFPI4F36 (“anti-TFPI”, mAbTFPI 2021) (doses:0.5, 1, 2 mg/kg) or an isotype control antibody 35 minutes beforeinduction of bleeding. The bleeding was observed for 1 hour (3600 sec).

FIG. 18 shows the platelet number measured in individual animals,following stimulation with anti-FVIII antibody, administration of ananti-TFPI-antibody (“anti-TFPI ab”, MuTFPI4F36) and then made to bleed.This was carried out in a control haemophilia model and in the presenceof the murine anti-TFPI antibody 4F36 (MuTFPI4F36) as described herein.

FIG. 19 shows the plasma concentration of free HzTFPI4F36 (mAbTFPI 2021)in rabbits dosed with 20 mg/kg HzTFPI4F36 at 0 hrs. Cuticle bleedingexperiments were performed at 96 hrs (4 days), 168 hrs (7 days) and 240hrs (10 days). The dotted lines indicate the ‘effective concentration’range of HzTFPI4F36 as found in the dose-response study (see FIG. 17).

FIG. 20: Left panel: plasma HzTFPI4F36 (mAbTFPI 2021) (left axis:∘) andcuticle bleeding time (mean±SEM;┘). Right panel: plasma HzTFPI4F36(mAbTFPI 2021) (left axis ∘) and blood loss (mean±SEM;┘) in rabbits withantibody-induced haemophilia, when pre-treated with 20 mg/kg HzTFPI4F36(n=8) or isotype control antibody (n=12) at 4, 7 or 10 days beforeinduction of bleeding. The bleeding was observed for 1 hour (3600 sec).

FIG. 21 shows the plasma concentration levels after IV and SC HzTFPI4F36(mAbTFPI 2021) administration to monkeys. On the three lower plots twomonkeys were administered three doses of HzTFPI4F36 with two weeksinterval. On the lower left three doses of 2, 20 and 80 mg/kg wereadministered, on the lower middle three doses of 20, 80 and 160 mg/kgwere administered; on the lower right three doses of 80, 160 and 200mg/kg were dosed. On the upper left a single dose of 20 mg/kg wereadministered to three monkeys; on the upper right as single IV dose wereadministered to three monkeys. On plots the points represent individualobservations whereas the line represents the model fit.

FIG. 22 shows a simulation of 1 mg/kg HzTFPI4F36 (mAbTFPI 2021)administered SC daily. The solid horizontal line represents simulatedplasma concentration levels and the dotted horizontal line the upperefficacious concentration as deduced from the effect data.

FIG. 23 shows a simulation of 15 mg/kg HzTFPI4F36 (mAbTFPI 2021),administered intravenously every third week. The solid horizontal linerepresents simulated plasma concentration levels and the dottedhorizontal line the upper efficacious concentration as deduced from theeffect data.

FIG. 24 shows a simulation of 20 mg/kg HzTFPI4F36 (mAbTFPI 2021),administered intravenously every second week. The horizontal solid linerepresents simulated plasma concentration levels and the horizontaldotted line the expected target saturation as deduced from the effectstudy.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 gives the amino acid sequence of human TFPI (signal peptidesequence omitted).

SEQ ID NO: 2 gives the amino acid sequence of a construct used fordetermining the binding epitope of an antibody. The construct comprisesamino acids 91 to 150 from human TFPI and a C-terminal His₆ tag.

SEQ ID NOs: 3, 5 and 4 give the polynucleotide (sense and anti-sense)and polypeptide sequences for the light chain variable domain (VL) ofthe MuTFPI4F36 (TFPI-4F36A1B2) monoclonal antibody. SEQ ID NO: 6 givesthe amino acid sequence of the light chain of the MuTFPI4F36(TFPI-4F36A1B2) monoclonal antibody. Signal peptide sequences areomitted.

SEQ ID NOs: 7, 9 and 8 give the polynucleotide (sense and anti-sense)and polypeptide sequences for the heavy chain variable domain (VH) ofthe MuTFPI4F36 (TFPI-4F36A1B2) monoclonal antibody. SEQ ID NO: 10 givesthe amino acid sequence of the heavy chain of the MuTFPI4F36(TFPI-4F36A1B2) monoclonal antibody. Signal peptide sequences areomitted.

SEQ ID NO: 11 gives the sequence of a reverse primer used for heavychain variable domain amplification and SEQ ID NO: 12 gives the sequenceof a reverse primer used for light chain amplification.

SEQ ID NOs: 13-15 provide the sense polynucleotide, anti-sensepolynucleotide and polypeptide sequences, respectively, for the lightchain variable domain (VL) of the humanized monoclonal antibody,HzTFPI4F36 (mAbTFPI2021). Signal peptide sequences are omitted.

SEQ ID NOs: 16-18 provide the sense polynucleotide, anti-sensepolynucleotide and polypeptide sequences, respectively, for the heavychain variable domain (VH) of the humanized monoclonal antibody,HzTFPI4F36 (mAbTFPI2021).

SEQ ID NOs: 19-21 provide the sense polynucleotide, anti-sensepolynucleotide and polypeptide sequences, respectively, for the lightchain (LC) of the humanized monoclonal antibody, HzTFPI4F36(mAbTFPI2021).

SEQ ID NOs: 22-24 provide the sense polynucleotide, anti-sensepolynucleotide and polypeptide sequences, respectively, for the heavychain (HC) of the humanized monoclonal antibody, HzTFPI4F36(mAbTFPI2021). Signal peptide sequences are omitted.

SEQ ID NOs: 25-26 provide the nucleic acid and amino acid sequences,respectively, for the light chain variable domain of the CDR-graftedHzTFPI4F36. Signal peptide sequences are omitted.

SEQ ID NOs: 27-28 provide the nucleic acid and amino acid sequences,respectively, of the heavy chain variable domain of the CDR-graftedHzTFPI4F36. Signal peptide sequences are omitted.

SEQ ID NO: 29 provides the amino acid sequence of the light chain of theCDR-grafted HzTFPI4F36 (human kappa chain). The signal peptide sequenceis omitted.

SEQ ID NO: 30 provides the amino acid sequence of the heavy chain of theCDR-grafted HzTFPI4F36, which is a human IgG4 (S241P). The signalpeptide sequence is omitted.

SEQ ID NO: 31 provides the germline sequence, VKII_A18/JK4, used forhumanization of the light chain of MuTFPI4F36. The signal peptidesequence is omitted.

SEQ ID NO: 32 provides the germline sequence, VH3_(—)21/JH6, used forhumanization of the heavy chain of MuTFPI4F36. The signal peptidesequence is omitted.

SEQ ID NO: 33 provides the amino acid sequence of the MuTFPI4F36A1B2heavy chain Fab. The signal peptide is omitted.

SEQ ID NO: 34 provides the amino acid sequence of the HzTFPI4F36 heavychain Fab. The signal peptide is omitted.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to antibodies that bind to TFPI. Theantibodies preferably specifically bind to TFPI, i.e. they bind to TFPIbut they do not bind, or bind at a lower affinity, to other molecules.In particular, the invention relates to antibodies that bind to TFPI andthat modulate its activity. Antibodies of the invention may thus possessthe ability to shorten clotting time. For example, an antibody of theinvention may have the ability to shorten clotting time in humanFVIII-deficient plasma or to reduce time to clot as measured in athromboelastography (TEG) analysis of human whole blood. The inventionalso relates to uses for such antibodies, such as therapeutic andpharmaceutical uses.

The term TFPI as used herein encompasses any naturally occurring form ofTFPI which may be derived from any suitable organism. For example, TFPIfor use as described herein may be a mammalian TFPI, such as human,mouse, rat, primate, bovine, ovine, or porcine TFPI. Preferably the TFPIis human TFPI. The TFPI may be a mature form of TFPI such as a TFPIprotein that has undergone post-translational processing within asuitable cell. Such a mature TFPI protein may, for example, beglycosylated. The TFPI may be a full length TFPI protein. The term TFPIalso encompasses variants, isoforms and other homologs of such TFPImolecules. Variant TFPI molecules will generally be characterised byhaving the same type of activity as naturally occurring TFPI, such asthe ability to neutralize the catalytic activity of FXa, or the abilityto inhibit a complex of TF-FVIIa/FXa.

An antibody of the invention will have the ability to bind to TFPI.Preferably, an antibody of the invention will bind specifically to TFPI.That is, an antibody of the invention will preferably bind to TFPI withgreater binding affinity than that at which it binds to anothermolecule. An antibody of the invention may have the ability to bind orspecifically bind to a TFPI molecule as described herein such as anytarget molecule as described herein.

The term “binding affinity” is herein used as a measure of the strengthof a non-covalent interaction between two molecules, e.g. and antibody,or fragment thereof, and an antigen. The term “binding affinity” is usedto describe monovalent interactions (intrinsic activity).

Binding affinity between two molecules, e.g. an antibody, or fragmentthereof, and an antigen, through a monovalent interaction may bequantified by determination of the dissociation constant (K_(D)). Inturn, K_(D) can be determined by measurement of the kinetics of complexformation and dissociation, e.g. by the SPR method (Biacore). The rateconstants corresponding to the association and the dissociation of amonovalent complex are referred to as the association rate constantsk_(a) (or k_(on)) and dissociation rate constant k_(d). (or k_(off)),respectively. K_(D) is related to k_(a) and k_(d) through the equationK_(D)=k_(d)/k_(a).

Following the above definition binding affinities associated withdifferent molecular interactions, e.g. comparison of the bindingaffinity of different antibodies for a given antigen, may be compared bycomparison of the K_(D) values for the individual antibody/antigencomplexes.

Similarly, the specificity of an interaction may be assessed bydetermination and comparison of the K_(D) value for the interaction ofinterest, e.g. a specific interaction between an antibody and anantigen, with the K_(D) value of an interaction not of interest.

Typically, the K_(D) for the antibody with respect to the target will be2-fold, preferably 5-fold, more preferably 10-fold less than K_(D) withrespect to the other, non-target molecule such as unrelated material oraccompanying material in the environment. More preferably, the K_(D)will be 50-fold less, such as 100-fold less, or 200-fold less; even morepreferably 500-fold less, such as 1,000-fold less, or 10,000-fold less.

The value of this dissociation constant can be determined directly bywell-known methods, and can be computed even for complex mixtures bymethods such as those, for example, set forth in Caceci et al. (Byte9:340-362, 1984). For example, the K_(D) may be established using adouble-filter nitrocellulose filter binding assay such as that disclosedby Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Otherstandard assays to evaluate the binding ability of ligands such asantibodies towards targets are known in the art, including for example,ELISAs, Western blots, RIAs, and flow cytometry analysis. The bindingkinetics and binding affinity of the antibody also can be assessed bystandard assays known in the art, such as Surface Plasmon Resonance(SPR), e.g. by using a Biacore™ system.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother ligand of that target, such as another antibody. Theconcentration at which 50% inhibition occurs is known as the Ki. Underideal conditions, the Ki is equivalent to K_(D). The Ki value will neverbe less than the K_(D), so measurement of Ki can conveniently besubstituted to provide an upper limit for K_(D).

An antibody of the invention may have a K_(D) for its target of 1×10⁻⁷Mor less, 1×10⁻⁸M or less, or 1×10⁻⁹M or less, or 1×10⁻¹⁰M or less,1×10⁻¹¹M or less, or 1×10⁻¹²M or less.

An antibody that specifically binds its target may bind its target witha high affinity, that is, exhibiting a low K_(D) as discussed above, andmay bind to other, non-target molecules with a lower affinity. Forexample, the antibody may bind to non-target molecules with a K_(D) of1×10⁻⁶M or more, more preferably 1×10⁻⁵ M or more, more preferably1×10⁻⁴ M or more, more preferably 1×10⁻³ M or more, even more preferably1×10⁻² M or more. An antibody of the invention is preferably capable ofbinding to its target with an affinity that is at least two-fold,10-fold, 50-fold, 100-fold 200-fold, 500-fold, 1,000-fold or 10,000-foldor greater than its affinity for binding to another non-target molecule.

The target molecule may be any TFPI molecule as described herein, suchas a naturally occurring TFPI molecule, a fully mature TFPI molecule ora full-length TFPI molecule. Preferred TFPI molecules are fully mature,naturally occurring, full length mammalian TFPI molecules. For example,the TFPI molecule may consist of, or may comprise, the amino acidsequence of SEQ ID NO: 1 or a fragment or other variant thereof asdescribed herein.

The target molecule may be a variant of a TFPI molecule such as afragment of a TFPI molecule. For example, the target molecule may be afragment or other variant of TFPI which maintains a suitable epitope forantibody binding. For example, the target molecule may be a fragment orother variant of TFPI which retains an epitope as described herein. Thetarget molecule may comprise such an epitope.

In one embodiment, the target molecule is a full length TFPI molecule.The full length TFPI molecule may comprise a first, second and thirdKunitz domain as described herein. The full length TFPI molecule maycomprise a first, second and third Kunitz domain as described herein andalso a carboxy terminal region as described herein. The full length TFPImolecule may be a naturally occurring TFPI molecule such as a fulllength TFPI polypeptide as expressed from a TFPI gene, or as secreted byTFPI expressing cells. The full length TFPI molecule may be a naturallyoccurring TFPI molecule as found circulating in free form in plasma orbound to cells such as endothelial cells. The full length TFPI moleculeis not a truncated TFPI molecule such as a naturally-occurring truncatedTFPI molecule as described herein.

In one embodiment, the target molecule is a truncated TFPI molecule. Forexample, the truncated TFPI molecule may comprise a carboxy terminaltruncation. For example, a number of naturally-occurring truncated formsof TFPI are known. These may comprise a truncation of part or all of thecarboxy terminal part of TFPI. They may further comprise truncation ofpart or all of one or more of the Kunitz domains. For example, atruncated form of TFPI may comprise the deletion of the carboxy terminalpart and part, or all, of the third Kunitz domain.

For example, one naturally occurring truncated form of TFPI comprisesonly amino acids 1 to 161 of the full length TFPI molecule (referred toherein as TFPI (1-161)). TFPI (1-161) is an active form of TFPI that hasreduced activity compared with the full length molecule. TFPI (1-161)differs in structure from full length TFPI and antibodies generatedagainst TFPI (1-161) as a target molecule may therefore differ fromantibodies generated against full length TFPI.

A truncated form of TFPI may be an appropriate target molecule where itis desired to target antibodies against the region of full length TFPIthat is present in TFPI (1-161). However, truncated TFPI is preferablyused as a target molecule when antibodies are desired to be directedagainst specific truncated forms of TFPI such as naturally occurringtruncated TFPI.

In one embodiment the target molecule is a naturally-occurring form ofTFPI. This may be used in a form in which it is present in vivo. Forexample, the target molecule may be a full length naturally-occurringTFPI as discussed above. The target molecule may be a truncatednaturally-occurring TFPI as discussed above. The target molecule may beTFPI in a form in which it is present in plasma in vivo. The targetmolecule may be TFPI that is bound to lipoprotein in the same way as ispresent in plasma in vivo. The target molecule may be TFPI that is boundto cells in the same way as occurs in vivo, such as TFPI that is boundto endothelial cells. An antibody of the invention may bind to any oneor more of these naturally occurring forms of TFPI. The antibody of theinvention may be able to bind to all of these naturally occurring formsof TFPI, or may be able to discriminate between these different forms,binding to some but not others.

In one embodiment, the target molecule is, or comprises, the secondKunitz domain of TFPI. Such a target molecule may comprise amino acids97 to 147 of SEQ ID NO: 1 or amino acids 91 to 150 of SEQ ID NO: 1 or anequivalent Kunitz domain 2 region from another TFPI polypeptide. Such atarget molecule may comprise SEQ ID NO: 2 or amino acids 3 to 58 or 10to 50 of SEQ ID NO: 2. The target molecule may be, or may comprise, afragment of the second Kunitz domain of TFPI. For example, the targetmolecule may comprise five or more, eight or more, ten or more, twelveor more or fifteen or more amino acids from the second Kunitz domain.

The target molecule may comprise five or more, eight or more, ten ormore, twelve or more or fifteen or more surface accessible residues ofTFPI or of a particular region of TFPI such as a particular Kunitzdomain or the C terminal part of TFPI. A surface accessible residue is aresidue having more than 40% relative accessibility. For example, forthe Kunitz 2 domain of TFPI (SEQ ID NO: 1), the following amino acidshave a greater than 40% relative accessibility: 94-95, 98, 100-110,118-121, 123-124, 131, 134, 138-142 and 144-145 (see FIG. 5). The targetmolecule may comprise five or more, eight or more, ten or more, twelveor more or fifteen or more of these residues, such as a fragment of TFPIthat includes five or more, eight or more, ten or more, twelve or moreor fifteen or more of these residues.

The target molecule may comprise a known epitope from TFPI. The term“epitope”, as used herein, is defined in the context of a molecularinteraction between an “antigen binding polypeptide” (Ab) and itscorresponding “antigen” (Ag). As used herein, the term Ab comprises anantibody or a fragment thereof, which specifically binds thecorresponding Ag. Examples of antigen-binding fragments include Fab,Fab′, F(ab)2, F(ab′)2, F(ab)S, Fv (typically the VL and VH domains of asingle arm of an antibody), single-chain Fv (scFv; see e.g. Bird et al.,Science 1988; 242:42 S-426; and Huston et al. PNAS 1988; 85:5879-5883),dsFv, Fd (typically the VH and CH1 domain), and dAb (typically a VHdomain) fragments; VH, VL, VhH, and V-NAR domains; monovalent moleculescomprising a single VH and a single VL chain; minibodies, diabodies,triabodies, tetrabodies, and kappa bodies (see, e.g., Ill et al. ProteinEng 1997; 10:949-57); camel IgG; IgNAR; as well as one or more isolatedCDRs or a functional paratope, where the isolated CDRs orantigen-binding residues or polypeptides can be associated or linkedtogether so as to form a functional antibody fragment. Various types ofantibody fragments have been described or reviewed in, e.g., Holligerand Hudson, Nat Biotechnol 2005; 25:1126-1136; WO2005040219, andpublished U.S. Patent Applications 20050238646 and 20020161201.

Antibody fragments can be obtained using conventional recombinant orprotein engineering techniques, and the fragments can be screened forantigen-binding or other function in the same manner as are can beintact antibodies.

The term antigen (Ag) refers to the molecular entity used forimmunization of an immunocompetent vertebrate to produce the antibody(Ab) that recognizes the Ag. Herein, Ag is termed more broadly and isgenerally intended to include target molecules that are specificallyrecognized by the Ab, thus including fragments or mimics of the moleculeused in the immunization process for raising the Ab. Thus, for Ab'sbinding to the second kunitz domain (K2) of TFPI, both isolated K2,full-length TFPI including truncated and other variants of TFPI arereferred to as an Ag.

Generally, the term “epitope” refers to the area or region on an Ag towhich an Ab specifically binds, i.e. the area or region in physicalcontact with the Ab. A protein epitope may comprise amino acid residuesin the Ag that are directly involved in binding to a Ab (also called theimmunodominant component of the epitope) and other amino acid residues,which are not directly involved in the binding, such as amino acidresidues of the Ag which are effectively blocked by the Ab (in otherwords, the amino acid residue is within the “solvent-excluded surface”and/or the “footprint” of the Ab). The term epitope herein includes bothtypes of binding sites in any particular region of K2 in TFPI thatspecifically binds to an anti-TFPI antibody, or another K2-specificagent according to the invention, unless otherwise stated (e.g., in somecontexts the invention relates to antibodies that bind directly toparticular amino acid residues). K2 may comprise a number of differentepitopes, which may include, without limitation, (1) linear peptideantigenic determinants, (2) conformational antigenic determinants whichconsist of one or more non-contiguous amino acids located near eachother in the mature K2 conformation; and (3) post-translationalantigenic determinants which consist, either in whole or part, ofmolecular structures covalently attached to K2, such as carbohydrategroups.

The epitope for a given antibody (Ab)/antigen (Ag) pair can be definedand characterized at different levels of detail using a variety ofexperimental and computational epitope mapping methods. The experimentalmethods include mutagenesis, X-ray crystallography, Nuclear MagneticResonance (NMR) spectroscopy, Hydroged deuterium eXchange MassSpectrometry (HX-MS) and various competition binding methods. As eachmethod relies on a unique principle the description of an epitope isintimately linked to the method by which it has been determined Thus,the epitope for a given Ab/Ag pair will be defined differently dependingon the epitope mapping method employed.

At its most detailed level, the epitope for the interaction between theAg and the Ab can be defined by the spatial coordinates defining theatomic contacts present in the Ag-Ab interaction, as well as informationabout their relative contributions to the binding thermodynamics. At aless detailed level the epitope can be characterized by the spatialcoordinates defining the atomic contacts between the Ag and Ab. At afurther less detailed level the epitope can be characterized by theamino acid residues that it comprises as defined by a specificcriterium, e.g. distance between atoms in the Ab and the Ag. At afurther less detailed level the epitope can be characterized throughfunction, e.g. by competition binding with other Abs. The epitope canalso be defined more generically as comprising amino acid residues forwhich substitution by another amino acid will alter the characteristicsof the interaction between the Ab and Ag.

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term epitope is herein, unless otherwise specified or contradictedby context, specifically defined as K2 residues characterized by havinga heavy atom (i.e. a non-hydrogen atom) within a distance of 4 Å from aheavy atom in the Ab.

From the fact that descriptions and definitions of epitopes, dependanton the epitope mapping method used, are obtained at different levels ofdetail, it follows that comparison of epitopes for different Abs on thesame Ag can similarly be conducted at different levels of detail.

Epitopes described on the amino acid level, e.g. determined from anX-ray structure, are said to be identical if they contain the same setof amino acid residues. Epitopes are said to overlap if at least oneamino acid is shared by the epitopes. Epitopes are said to be separate(unique) if no amino acid residue are shared by the epitopes.

Epitopes characterized by competition binding are said to be overlappingif the binding of the corresponding Ab's are mutually exclusive, i.e.binding of one Ab excludes simultaneous binding of the other Ab. Theepitopes are said to be separate (unique) if the Ag is able toaccommodate binding of both corresponding Ab's simultaneously.

The definition of the term “paratope” is derived from the abovedefinition of “epitope” by reversing the perspective. Thus, the term“paratope” refers to the area or region on the Ab to which an Agspecifically binds, i.e. to which it makes physical contact to the Ag.

In the context of an X-ray derived crystal structure defined by spatialcoordinates of a complex between an Ab, e.g. a Fab fragment, and its Ag,the term paratope is herein, unless otherwise specified or contradictedby context, specifically defined as Ag residues characterized by havinga heavy atom (i.e. a non-hydrogen atom) within a distance of 4 Å from aheavy atom in K2.

The epitope and paratope for a given antibody (Ab)/antigen (Ag) pair maybe identified by routine methods. For example, the general location ofan epitope may be determined by assessing the ability of an antibody tobind to different fragments or variant TFPI polypeptides. The specificamino acids within TFPI that make contact with an antibody (epitope) andthe specific amino acids in an antibody that make contact with TFPI(paratope) may also be determined using routine methods, such as thosedescribed in the examples. For example, the antibody and target moleculemay be combined and the Ab/Ag complex may be crystallised. The crystalstructure of the complex may be determined and used to identify specificsites of interaction between the antibody and its target.

The present inventors have carried out such an analysis for theinteraction between the murine MuTFPI4F36 antibody, as well as thehumanised HzTFPI4F36 antibody, described herein, and the Kunitz 2 domain(K2) of TFPI. This analysis is described in more detail in the examples.

The paratope of an antibody according to the current invention may bedefined as follows: the light chain of said antibody comprises residuesE31, S32, D33, Y37, A96, T97, and F99 of SEQ ID NO: 15 and the heavychain of said antibody comprises residues N31, S52, R53, S54, Y57, Y59,F60, P61, D62, Q65, Y102, D103 and D106 of SEQ ID NO 18.

The light chain of the antibody according to the current invention maythus comprise amino acid residues:

-   -   E, in the position corresponding to position 31,    -   S, in the position corresponding to position 32,    -   D, in the position corresponding to position 33,    -   Y, in the position corresponding to position 37,    -   A, in the position corresponding to position 96,    -   T, in the position corresponding to position 97 and    -   F, in the position corresponding to position 99 of SEQ ID NO:        15; and the heavy chain of said antibody may comprise amino acid        residues:    -   N, in the position corresponding to position 31,    -   R, in the position corresponding to position 53,    -   S, in the position corresponding to position 54,    -   Y, in the position corresponding to position 57,    -   Y, in the position corresponding to position 59,    -   F, in the position corresponding to position 60,    -   P, in the position corresponding to position 61,    -   D, in the position corresponding to position 62,    -   Q, in the position corresponding to position 65,    -   Y, in the position corresponding to position 102,    -   D, in the position corresponding to position 103 and    -   D, in the position corresponding to position 106 of SEQ ID NO        18.

The heavy chain may further comprise an S in the position correspondingto position 52 of SEQ ID NO: 18.

The light chain of an antibody according to the current invention mayfurther comprise an H in the position corresponding to position 98 ofSEQ ID NO: 15 and the heavy chain may further comprise an S, in theposition corresponding to position 56 of SEQ ID NO: 18.

For MuTFPI4F36 (Example 4) the epitope was found to be composed of aminoacids E100, E101, P103, R107, Y109, T111, Y113, Q118, Q121, E123, R124,F125, K126 and L140 of SEQ ID NO: 1, corresponding to amino acids E10,E11, P13, R17, Y19, T21, Y23, Q28, Q31, E33, R34, F35, K36 and L50 ofSEQ ID NO: 2. The paratope was found to be composed of light chain aminoacid residues E31, S32, D33, Y37, A96, T97, H98 and F99 of SEQ ID NO: 4and the heavy chain amino acid residues N31, R53, S54, S56, Y57, Y59,F60, P61, D62, Q65, Y102, D103 and D106 of SEQ ID NO 8.

For HzTFPI4F36 (Example 5) the epitope was found to be composed of aminoacids E100, E101, D102, P103, R107, Y109, T111, Y113, F114, N116, Q118,Q121, C122, E123, R124, F125, K126 and L140 of SEQ ID NO: 1,corresponding to amino acids E10, E11, D12, P13, R17, Y19, T21, Y23,F24, N26, Q28, Q31, C32, E33, R34, K36 and L50 of SEQ ID NO: 2. Theparatope was found to be composed of light chain amino acid residuesE31, S32, D33, Y37, A96, T97 and F99 of SEQ ID NO: 15 and the heavychain amino acid residues N31, S52, R53, S54, Y57, Y59, F60, P61, D62,Q65, Y102, D103 and D106 of SEQ ID NO 18.

An antibody according to the current invention may bind to the sameepitope or domain of TFPI as the antibodies of the invention that arespecifically disclosed herein. For example, other yet unidentifiedantibodies of the invention may be identified by comparing their bindingto TFPI with that of the monoclonal antibodies, MuTFPI4F36 and/orHzTFPI4F36; or by comparing the function of yet unidentified antibodieswith that of MuTFPI4F36 and/or HzTFPI4F36. Analyses and assays that maybe used for the purpose of such identification include TFPI neutralizingassays such as: the FXa inhibition assay described in example 6 and theFVIIa/TF/FXa inhibition assay described in example 7; bindinginteraction analyses such as the surface plasmon resonance analysisdescribed in example 8; cellular assays such as the neutralization ofTFPI on human umbilical vascular endothelial cells (HUVECs), describedin example 9, and the neutralization of TFPI inhibition of TF/FVIIaactivity on MDA-MB 231 human breast carcinoma cells, described inexample 10.

In one embodiment, an antibody of the invention may bind to the sameepitope or region as the MuTFPI4F36 or HzTFPI4F36 antibodies describedherein. The binding of MuTFPI4F36 and HzTFPI4F36 to TFPI is described inmore detail herein. An antibody of the invention may be an antibody thatbinds to the same epitope in TFPI as the MuTFPI4F36 or HzTFPI4F36antibodies. This may include it being in contact with the particularamino acids of TFPI as described above. For example, an antibody of theinvention may bind to TFPI in such a way that it is in contact withamino acids E10, E11, P13, R17, Y19, T21, Y23, Q28, Q31, E33, R34, F35,K36 and L50 of SEQ ID NO: 2. or in such a way that it is in contact withamino acids E10, E11, D12, P13, R17, Y19, T21, Y23, F24, N26, Q28, Q31,C32, E33, R34, K36 and L50 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising one or more residues selected from the group consisting ofE10, E11, D12, P13, R17, Y19, T21, Y23, F24, N26, Q28, Q31, C32, E33,R34, F35, K36 and L50 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue E10 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue E11 of SEQ ID NO: 2).

An antibody of the invention may be capable of binding an epitopecomprising residue D12 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue P13 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue R17 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue Y19 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue T21 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue Y23 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue F24 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue N26 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue Q28 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue Q31 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue C32 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue E33 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue R34 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue F35 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue K36 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residue L50 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residues E10, E11, D12, P13, R17, Y19, T21, Y23, F24, N26,Q28, Q31, C32, E33, R34, K36 and L50 of SEQ ID NO: 2.

An antibody of the invention may be capable of binding an epitopecomprising residues E10, E11, P13, R17, Y19, T21, Y23, Q28, Q31, E33,R34, F35, K36 and L50 of SEQ ID NO: 2.

An antibody of the invention may have the ability to compete withanother antibody of the invention for binding to TFPI or anotherappropriate target as described herein. For example, an antibody of theinvention may cross-compete with the MuTFPI4F36 or HzTFPI4F36 antibodiesdescribed herein for binding to TFPI, or to a suitable fragment orvariant of TFPI that is bound by the MuTFPI4F36 or HzTFPI4F36antibodies. Such cross-competing antibodies can be identified based ontheir ability to cross-compete with a known antibody of the invention instandard binding assays. For example, SPR e.g. by using a Biacore™system, ELISA assays or flow cytometry may be used to demonstratecross-competition. Such cross-competition may suggest that the twoantibodies bind to identical, overlapping or similar epitopes.

Thus, the antibody of the invention may be capable of binding the K2domain of TFPI with a higher affinity than any one or more of thefollowing commercially available monoclonal antibodies: mAb0281 (Absystems) and/or mAb4904 (American Diagnostica) and/or mAb2974 (R&Dsystems) and/or mAb29741 (R&D systems).

An antibody of the invention may therefore be identified by a methodthat comprises a binding assay which assesses whether or not a testantibody is able to compete with a known antibody of the invention for abinding site on the target molecule. Methods for carrying outcompetitive binding assays are well known in the art. For example theymay involve binding a known antibody of the invention to a targetmolecule using conditions under which the antibody can bind to thetarget molecule. The antibody/target complex may then be exposed to atest antibody and the extent to which the test antibody is able todisplace the antibody of the invention from antibody/target complexesmay be assessed. An alternative method may involve contacting a testantibody with a target molecule under conditions that allow for antibodybinding, then adding an antibody of the invention that is capable ofbinding that target molecule and assessing the extent to which theantibody of the invention is able to displace the test antibody fromantibody/target complexes.

The ability of a test antibody to inhibit the binding of an antibody ofthe invention to the target demonstrates that the test compound cancompete with an antibody of the invention for binding to the target andthus that the test antibody binds to the same epitope or region on theTFPI protein as the known antibody of the invention. A test antibodythat is identified as competing with a known antibody of the inventionin such a method is also a potential antibody according to the presentinvention. The fact that the test antibody can bind TFPI in the sameregion as a known antibody of the invention and compete with the knownantibody of the invention suggests that the test antibody may act as aligand at the same binding site as the known antibody and that the testantibody may therefore mimic the action of the known antibody. This canbe confirmed by assessing the activity of TFPI in the presence of thetest compound as described herein.

The known antibody of the invention may be an antibody as describedherein, such as the murine TFPI-4F36A1B2 (also referred to as 4F36 andas MuTFPI4F36) antibody, or any variant or fragment thereof as describedherein that retains the ability to bind to TFPI, such as humanizedTFPI-4F36A1B2 antibodies, one of which is herein referred to asHzTFPI4F36 (mAbTFPI 2021). An antibody of the invention may bind to thesame epitope as the MuTFPI4F36 antibody as described herein or anyvariant or fragment thereof as described herein that retains the abilityto bind to TFPI, such as HzTFPI4F36.

An antibody of the invention may bind an epitope that is identical to,overlaps, or is similar to the MuTFPI4F36 epitope that is furtherdescribed in the examples. An antibody of the invention may bind to anepitope that is identical to, overlaps or is similar to the HzTFPI4F36epitope that is further described in the examples. An antibody of theinvention may bind, preferably specifically, one or more amino acidresidues that belong to the epitopes of MuTFPI4F36 and/or HzTFPI4F36.For example, an antibody of the invention may bind to five or more, sixor more, seven or more, eight or more or ten or more of the amino acidresidues set out above for binding of MuTFPI4F36 or HzTFPI4F36. Forexample, when contacted with a polypeptide of SEQ ID NO: 2, an antibodyof the invention may bind to the polypeptide and make contact with aminoacids E10, E11, D12, P13, R17, Y19, T21, Y23, F24, N26, Q28, Q31, C32,E33, R34, F35, K36 and L50, or a subset of those amino acids, such as atleast 2, at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, at least 10, at least 11, at least 12, at least 13,at least 14, at least 15, at least 16, at least 17 or at least 18 ofthose amino acids.

Specific binding may be assessed with reference to binding of theantibody to a molecule that is not the target. This comparison may bemade by comparing the ability of an antibody to bind to the target andto another molecule. This comparison may be made as described above inan assessment of K_(D) or Ki. The other molecule used in such acomparison may be any molecule that is not the target molecule.Preferably the other molecule is not identical to the target molecule.Preferably the target molecule is not a fragment of the target molecule.

The K_(D) of an antibody of the current invention may be less than 0.8nM, such as less than 0.7 nM, such as less than 0.6 nM, such as lessthan 0.5 nM, such as less than 0.4 nM, such as less than 0.3 nM, such asless than 0.2 nM, such as less than 0.1 nM, such as less than 0.05 nM,such as less than 0.025 nM, such as less than 0.015 nM, such as between0.015 nM and 0 nM.

The other molecule used to determine specific binding may be unrelatedin structure or function to the target. For example, the other moleculemay be an unrelated material or accompanying material in theenvironment.

The other molecule used to determine specific binding may be anothermolecule involved in the same in vivo pathway as the target molecule.For example, where the target is TFPI or a fragment or variant thereof,the other molecule used for comparison may be a protein that forms partof the blood coagulation cascade. By ensuring that the antibody of theinvention has specificity for TFPI over another such molecule, unwantedin vivo cross-reactivity may be avoided.

The other molecule used for comparison may be related to the targetmolecule. For example, where it is desired to identify an antibody thatbinds only to a specific epitope, the other molecule for comparison maybe a TFPI molecule in which that epitope is lacking or disrupted. Theother molecule used for comparison may thus be another target moleculethat is different to the target molecule bound by the antibody inquestion.

The antibody of the invention may retain the ability to bind to somemolecules that are related to the target molecule. For example, afull-length mature human TFPI may be used as the target, but theantibody may also be able to bind to, e g immature forms of human TFPI,fragments or truncated forms of human TFPI, TFPI that is bound tolipoprotein or to a cell or TFPI from other species, such as othermammalian TFPI.

Alternatively, the antibody of the invention may have specificity for aparticular target molecule. For example, it may bind to one targetmolecule as described herein, but may not bind, or may bind withsignificantly reduced affinity to a different target molecule asdescribed herein. For example, a full length mature human TFPI may beused as the target, but the antibody that binds to that target may beunable to bind to or may bind with lesser affinity to, e.g. immatureforms of human TFPI, fragments or truncated forms of human TFPI, TFPIthat is bound to lipoprotein or to a cell or TFPI from other species,such as other mammalian TFPI.

An antibody of the invention may bind to TFPI and in doing so mayinhibit an activity of TFPI.

As explained above, TFPI downregulates blood coagulation. It does thisby inhibiting the activity of FXa and by inhibiting the TF-FVIIa complexin the presence of FXa. The activity of TFPI that is inhibited by anantibody of the invention may be any of these activities or anydownstream effect thereof. For example, an antibody of the invention maylead to an increase in blood coagulation, an increase in the presence orlevels of FXa or an increased activity of TF-FVIIa. Preferably, anantibody of the invention reduces clotting time when contacted with (a)human FVIII deficient plasma or (b) human whole blood.

The measurement of TFPI activity may comprise assessing the activity ofthe TFPI in inhibiting coagulation or reducing clotting time in a bloodsample. For example, such a method may comprise contacting TFPI with asample of blood or a blood product such as plasma or serum thatcomprises blood coagulation factors under conditions in whichcoagulation should occur, and determining whether coagulation of theblood is inhibited or clotting time is reduced by the presence of theTFPI. The level of blood coagulation or clotting time in such a samplemay then be compared to that in an equivalent sample in which a testantibody is also present. If the level of coagulation is increased orclotting time is reduced in the antibody sample, this suggests that theantibody is inhibiting the activity of TFPI in the sample.

Blood coagulation may be detected by looking for coagulation of theblood itself, of plasma, or for one or more characteristics of thecoagulation cascade that lie downstream to the point of action of TFPI.For example, the method may assess levels of FXa or activation ofTF-FVIIa in the sample.

Various other methods for assessing blood coagulation and clotting timeare well known in the art. For example, any effect of an antibody onblood clotting time may be assessed using a dilute prothrombin timeanalysis (dPT analysis) as described in the examples. Briefly, humanplasma is contacted with human thromboplastin. The time taken for theplasma to clot is measured in the presence and absence of the testantibody. A positive control may be used in such an analysis, such asaddition of FVIIa (NovoSeven®) which would be expected to reduceclotting time. An antibody of the invention should be capable ofreducing clotting time in such a method. Preferably, an antibody of theinvention should be capable of reducing clotting time in adose-dependent manner.

The antibody of the current invention may be capable of inhibiting TFPIin a plasma-based clot assay, such as a dPT analysis, significantlybetter than any one or more of the following commercially availablemonoclonal antibodies: mAb0281 (Ab systems) and/or mAb4904 (AmericanDiagnostica) and/or mAb2974 (R&D systems) and/or mAb29741 (R&D systems).

Thromboelastography may be used to assess the kinetics of clot formationand fibrinolysis in samples of whole blood. The ability of an antibodyto reduce clotting time or to stimulate blood coagulation may thus besimilarly assessed in a whole blood sample by comparing the time takenfor clot formation in the presence and absence of the antibody.

Methods to assess the functional effects of an antibody of the inventionmay thus be carried out in vitro. Such methods are preferably carriedout on samples of human blood or plasma. Such samples may be normalhuman blood or plasma or may be deficient in, or supplemented with, oneor more factors involved in blood coagulation. For example, thesemethods may be carried out using normal human whole blood, normal humanplasma or FVIII-deficient plasma or whole blood. FVIII-deficient bloodor plasma may be generated by contacting a suitable blood or plasmasample with neutralising anti-FVIII antibody. Such in vitro methods maybe binding interaction analyses or TFPI neutralisation analyses, such asthose described in examples 6-11.

The antibody of the current invention may be capable of inhibitingplatelet-associated TFPI.

The antibody of the current invention may be capable of inhibitingsoluble TFPI. The antibody of the current invention may be capable ofinhibiting lipoprotein-bound TFPI.

The antibody of the current invention may be capable of inhibitingcell-bound TFPI, such as TFPI that is bound to endothelial cells.

The antibody of the current invention may be capable of binding TFPIsuch that FXa retains its activity by at least 91%, such as at least92%, such as at least 93%, such as at least 94%, such as at least 95%,such as at least 96%, such as at least 97%, such as at least 98%, suchas at least 99%, such as 99-100% as measured in a FXa inhibition assay.

The antibody of the current invention may be capable of neutralising theTFPI inhibition of membrane-bound FVIIa/TF/FXa, when TFPI is saturatedwith said antibody, by at least 55%, such as at least 60%, such as atleast 65%, such as at least 70%, such as at least 75%, such as at least80%, such as at least 85%, such as at least 90%, such as at least 95%,such as up to 100%, such as 100%, as measured in a FVIIa/TF/FXainhibitor assay.

Preferably, an antibody of the invention is capable of reducing clottingtime and/or stimulating blood coagulation in a sample of (a) human wholeblood, (b) human plasma, (c) FVIII-deficient human whole blood, (d)FVIII-deficient human plasma, (e) FIX-deficient human whole blood or (f)FIX-deficient human plasma.

Methods to determine the ability of an antibody to stimulate bloodcoagulation or reduce clotting time may also be carried out in vivo. Forexample, in vivo studies may be carried out in transient haemophilicrabbits as described in the examples. Briefly, rabbits may be madetransient haemophilic by administration of anti-FVIII antibody. The testantibody may then be administered and cuticle bleed time and/or plateletnumber assessed. A reduction in cuticle bleed time in the presence of atest antibody indicates that the antibody is capable of reducingclotting time and stimulating blood coagulation. An antibody having suchan effect may therefore be an antibody of the present invention.

The antibody of the current invention may be capable of binding the K2domain of TFPI such that the percentage of free TFPI in a subject isreduced to less than 30%, such as less than 29%, such as less than 28%,such as less than 27%, such as less than 26%, such as less than 25%,such as less than 24%, such as less than 23%, such as less than 22%,such as less than 21%, such as less than 20%, such as less than 19%,such as less than 18%, such as less than 17%, such as less than 16%,such as less than 15%, such as less than 14%, such as less than 13%,such as less than 12%, such as less than 11%, such as less than 10%,such as less than 9%, such as less than 8%, such as less than 7%, suchas less than 6%, such as less than 5%, such as less than 4%, such asless than 3%, such as less than 2%, such as less than 1%, such as 0%.

Furthermore, the antibody of the current invention may be capable ofbinding the K2 domain of TFPI such that the amount of free TFPI in asubject is reduced during the first 28 days, such as during the first 27days, such as during the first 26 days, such as during the first 25days, such as during the first 24 days, such as during the first 23days, such as during the first 22 days, such as during the first 21days, such as during the first 20 days, such as during the first 19days, such as during the first 18 days, such as during the first 17days, such as during the first 16 days, such as during the first 15days, such as during the first 14 days, such as during the first 13days, such as during the first 12 days, such as during the first 11days, such as during the first 10 days, such as during the first 9 days,such as during the first 8 days, such as during the first 7 days, suchas during the first 6 days, such as during the first 5 days, such asduring the first 4 days, such as during the first 3 days, such as duringthe first 2 days, such as during the first day after administration ofsaid monoclonal antibody to said subject.

An antibody of the present invention may also lead to no significantdecrease in platelet numbers. In particular, an antibody of theinvention may be capable of reducing clotting time and/or stimulatingblood coagulation in a sample of (a) human whole blood, (b) humanplasma, (c) FVIII-deficient human whole blood (d) FVIII-deficient humanplasma, (e) FIX-deficient human whole blood or (f) FIX-deficient humanplasma, or in an animal in vivo, without leading to any significantdecrease in platelet numbers. Platelet numbers can be assessed in thesame sample or animal as the other effects discussed above, or can beassessed separately. For example, platelet numbers can be assessed in ablood sample such as a sample of blood obtained from a patient orexperimental animal. Platelet numbers may be assessed followingadministration of the antibody to a transient haemophilic rabbit asdescribed above. Antibodies of the invention may be capable of reducingcuticle bleed time without leading to a concurrent decrease in plateletnumbers, as exemplified by in vivo studies in transient haemophilicrabbits. A change in platelet numbers may be assessed by comparingplatelet numbers before and after administration of the antibody or bycomparing platelet numbers between a sample or animal treated with theantibody of interest and a control sample or animal not treated withthat antibody. An antibody of the current invention may be capable ofbinding the K2 domain of TFPI, such that a subject's in vivo clottingtime is reduced and said subject's platelet count is not significantlyreduced. For example, said subject's platelet count may not fall toapproximately 80%, such as approximately 75%, such as approximately 70%,such as approximately 65%, such as approximately 60%, such asapproximately 55%, such as approximately 50%, such as approximately 45%,such as approximately 40%, such as approximately 35%, such asapproximately 30%, such as approximately 25% of the original plateletcount. Preferably, there will be no difference or no statisticallysignificant difference in platelet numbers when making such comparisons.That is, the antibody of the invention will not have caused any decreasein platelet numbers.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An antibody refers to a glycoprotein comprising at leasttwo heavy chains (HC) and two light chains (LC) inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region (CH). Each light chain is comprised ofa light chain variable region (abbreviated herein as VL) and a lightchain constant region (CL). The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. The VHand VL regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR). The constant regions of the antibodies may mediate thebinding of the immunoglobulin to host tissues or factors, includingvarious cells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

The term “complementarity-determining region” or “hypervariable region”when used herein refers to the amino acid residues of an antibody thatare responsible for antigen binding. The complementarity-determiningregions or “CDRs” are generally comprised of amino acid residues 24-34(L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variabledomain; (Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242) and/or those residues from a “hypervariableloop” (residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy-chain variable domain; Chothia and Lesk, J. Mol. Biol. 1987;196:901-917). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “Kabat residue”, and “according toKabat” herein refer to this numbering system for heavy chain variabledomains or light chain variable domains. Using the Kabat numberingsystem, the actual linear amino acid sequence of a peptide may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include amino acid insertions (residue 52a,52b and 52c according to Kabat) after residue 52 of CDR H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat)after heavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

The term “framework region” or “FR” residues refer to those VH or VLamino acid residues that are not within the CDRs, as defined herein.

An antibody of the invention may be a monoclonal antibody or apolyclonal antibody. In one embodiment, an antibody of the invention isa monoclonal antibody. An antibody of the invention may be a chimericantibody, a CDR-grafted antibody, a human or humanised antibody or anantigen binding portion of any thereof. For the production of bothmonoclonal and polyclonal antibodies, the experimental animal is asuitable a mammal such as, but not restricted to, a goat, rabbit, rat ormouse.

Polyclonal antibodies are antibodies that are derived from different Bcell lines. A polyclonal antibody may comprise a mixture of differentimmunoglobulin molecules that are directed against a specific antigen.The polyclonal antibody may comprise a mixture of differentimmunoglobulin molecules that bind to one or more different epitopeswithin an antigen molecule. Polyclonal antibodies may be produced byroutine methods such as immunisation of a suitable animal, with theantigen of interest. Blood may be subsequently removed from the animaland the immunoglobulin fraction purified.

Monoclonal antibodies are immunoglobulin molecules that are identical toeach other and have a single binding specificity and affinity for aparticular epitope. Monoclonal antibodies (mAbs) of the presentinvention can be produced by a variety of techniques, includingconventional monoclonal antibody methodology e.g., the standard somaticcell hybridization technique of Kohler and Milstein (1975) Nature 256:495, or viral or oncogenic transformation of B lymphocytes. Thepreferred animal system for preparing hybridomas is the murine system.Hybridoma production in the mouse is a very well-established procedure.Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known.

To generate hybridomas producing monoclonal antibodies of the invention,splenocytes and/or lymph node cells from immunized mice can be isolatedand fused to an appropriate immortalized cell line, such as a mousemyeloma cell line. The resulting hybridomas can be screened for theproduction of antigen-specific antibodies. The antibody secretinghybridomas can be replated, screened again, and if still positive forsuitable IgG, the monoclonal antibodies can be subcloned at least twiceby limiting dilution. The stable subclones can then be cultured in vitroto generate small amounts of antibody in tissue culture medium forcharacterization.

The term “antigen-binding portion” of an antibody refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen, such as TFPI or another target protein as described herein.It has been shown that the antigen-binding function of an antibody canbe performed by fragments of a full-length antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include a Fab fragment, a F(ab′)₂ fragment, a Fab′ fragment, aFd fragment, a Fv fragment, a dAb fragment and an isolatedcomplementarity determining region (CDR). Single chain antibodies suchas scFv and heavy chain antibodies such as VHH and camel antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. These antibody fragments may be obtained usingconventional techniques known to those of skill in the art, and thefragments may be screened for utility in the same manner as intactantibodies.

An antibody of the invention may be prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for theimmunoglobulin genes of interest or a hybridoma prepared therefrom, (b)antibodies isolated from a host cell transformed to express the antibodyof interest, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of immunoglobulin gene sequences to other DNA sequences.

An antibody of the invention may be a human antibody or a humanisedantibody. The term “human antibody”, as used herein, is intended toinclude antibodies having variable regions in which both the frameworkand CDR regions are derived from human germline immunoglobulinsequences. Furthermore, if the antibody contains a constant region, theconstant region also is derived from human germline immunoglobulinsequences. The human antibodies of the invention may include amino acidresidues not encoded by human germline immunoglobulin sequences (e.g.,mutations introduced by random or site-specific mutagenesis in vitro orby somatic mutation in vivo). However, the term “human antibody”, asused herein, is not intended to include antibodies in which CDRsequences derived from the germline of another mammalian species, suchas a mouse, have been grafted onto human framework sequences.

Such a human antibody may be a human monoclonal antibody. Such a humanmonoclonal antibody may be produced by a hybridoma which includes a Bcell obtained from a transgenic nonhuman animal, e.g., a transgenicmouse, having a genome comprising a human heavy chain transgene and alight chain transgene fused to an immortalized cell.

Human antibodies may be isolated from sequence libraries built onselections of human germline sequences further diversified with naturaland synthetic sequence diversity.

Human antibodies may be prepared by in vitro immunisation of humanlymphocytes followed by transformation of the lymphocytes withEpstein-Barr virus.

The term “human antibody derivatives” refers to any modified form of thehuman antibody, e.g., a conjugate of the antibody and another agent orantibody.

The term “humanized antibody” is intended to refer to a human/non-humanchimeric antibody that contains a minimal sequence (CDR regions) derivedfrom non-human immunoglobulin. Humanized antibodies are thus humanimmunoglobulins (recipient antibody) in which residues from ahyper-variable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit, or non-human primate hav-ing the desiredspecificity, affinity, and capacity. In some instances, FR residues ofthe human immunoglobulin are replaced by corresponding non-humanresidues. An example of such a modification is the introduction of oneor more so-called back-mutations, such as is described in example 2.

Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, a humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR residuesare those of a human immunoglobulin sequence. The humanized antibody canoptionally also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.

Antibodies of the invention can be tested for binding to the targetprotein by, for example, standard ELISA or Western blotting. An ELISAassay can also be used to screen for hybridomas that show positivereactivity with the target protein. The binding specificity of anantibody may also be determined by monitoring binding of the antibody tocells expressing the target protein, for example by flow cytometry.

The specificity of an antibody of the invention for target protein maybe further studied by determining whether or not the antibody binds toother proteins. For example, where it is desired to produce an antibodythat specifically binds TFPI or a particular part, e.g. epitope, ofTFPI, the specificity of the antibody may be assessed by determiningwhether or not the antibody also binds to other molecules or modifiedforms of TFPI that lack the part of interest.

As explained above, antibodies of the invention may modify the activityof TFPI. Antibodies having the required binding properties may thus befurther tested to determine their effects on the activity of TFPI. Thus,methods may be used to identify suitable antibodies that are capable ofbinding to TFPI and that are capable of modifying, and in particularreducing, its activity.

Once a suitable antibody has been identified and selected, the aminoacid sequence of the antibody may be identified by methods known in theart. The genes encoding the antibody can be cloned using specific and/ordegenerate primers. The antibody may be recombinantly produced byroutine methods.

A “polypeptide” is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The term “polypeptide” thus includes shortpeptide sequences and also longer polypeptides and proteins. As usedherein, the term “amino acid” may refer to natural and/or unnatural orsynthetic amino acids, D and/or L optical isomers, and amino acidanalogs and peptidomimetics.

The present inventors have identified a murine antibody as described inthe examples. This antibody is referred to herein as TFPI-4F36A1B2(alternatively, 4F36 or MuTFPI4F36). The present invention encompassesthis antibody, variants and fragments thereof—including chimericantibodies and humanised antibodies—which retain one or more activitiesof the murine antibody and which are also described in the examples. Theactivities of this antibody include the ability to bind to TFPI, theability to bind to specific locations in the TFPI molecule and theability to inhibit the activity of TFPI.

A suitable fragment or variant of this antibody will retain the abilityto bind to TFPI. It will preferably retain the ability to specificallybind to TFPI. It will preferably retain the ability to specifically bindto the same or similar epitope or region of the TFPI molecule as theantibody (MuTFPI4F36) from which it is derived. It will preferablyretain one or more additional functions of the antibody from which it isderived, such as the ability to inhibit TFPI activity or the ability toreduce clotting time, optionally without leading to a drop in plateletnumbers.

Polypeptide or antibody “fragments” according to the invention may bemade by truncation, e.g. by removal of one or more amino acids from theN and/or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30,up to 40 or more amino acids may be removed from the N and/or C terminalin this way. Fragments may also be generated by one or more internaldeletions.

An antibody of the invention may be, or may comprise, a fragment of theMuTFPI4F36 antibody or a variant thereof. The antibody of the inventionmay be or may comprise an antigen binding portion of this antibody or avariant thereof as discussed further above. For example, the antibody ofthe invention may be a Fab fragment of this antibody or a variantthereof or may be a single chain antibody derived from this antibody ora variant thereof.

The amino acid sequences of the light and heavy chains of the MuTFPI4F36antibody are given in SEQ ID NOs: 6 and 10 respectively. The amino acidsequences for the VL and VH chains of the MuTFPI4F36 antibody are givenin SEQ ID NOs: 4 and 8 respectively. The amino acid sequences of thelight and heavy chains of one humanised antibody, HzTFPI4F36, are givenin SEQ ID NOs: 21 and 24, respectively. The amino acid sequences for theVL and VH chains of HzTFPI4F36 are given in SEQ ID NOs: 15 and 18,respectively.

An antibody of the invention may comprise the MuTFPI4F36 light chainamino acid sequence shown in SEQ ID NO: 6 or a fragment or variantthereof. An antibody may additionally or alternatively comprise theMuTFPI4F36 heavy chain amino acid sequence shown in SEQ ID NO: 10 or afragment or variant thereof as described herein.

An antibody of the invention may comprise the VL amino acid sequence ofSEQ ID No: 4, or a fragment or variant thereof. An antibody of theinvention may comprise the VH amino acid sequence of SEQ ID No: 8, or afragment or variant thereof. An antibody of the invention may compriseboth (a) the VL amino acid sequence of SEQ ID No: 4, or a fragment orvariant thereof and (b) the VH amino acid sequence of SEQ ID No: 8, or afragment or variant thereof.

An antibody of the invention may comprise a fragment of one of the VL orVH amino acid sequences shown in FIG. 2. For example, an antibody of theinvention may comprise a fragment of at least 7, at least 8, at least 9,at least 10, at least 12, at least 15, at least 18, at least 20 or atleast 25 consecutive amino acids from SEQ ID No: 4 or 8. Such a fragmentwill preferably retain one or more of the functions discussed above,such as the ability to bind to TFPI.

A suitable fragment or variant of any of these VH or VL sequences willretain the ability to bind to TFPI. It will preferably retain theability to specifically bind to TFPI. It will preferably retain theability to specifically bind to the same or similar epitope or region ofthe TFPI molecule as the antibody (MuTFPI4F36) from which it is derived.It will preferably retain one or more additional functions of theantibody from which it is derived, such as the ability to inhibit TFPIactivity or the ability to reduce clotting time, optionally withoutleading to a drop in platelet numbers.

A suitable fragment or variant VL sequence will preferably retain theamino acids at positions E31, S32, D33, Y37, A96, T97, H98 and F99 inSEQ ID NO: 4. A suitable fragment or variant VH sequence will preferablyretain the amino acids at positions N31, R53, S54, S56, Y57, Y59, F60,P61, D62, Q65, Y102, D103 and D106 in SEQ ID NO: 8. A suitable fragmentor variant antibody will preferably retain the amino acids at positionsE31, S32, D33, Y37, A96, T97, H98 and F99 in SEQ ID NO: 4 and the aminoacids at positions N31, R53, S54, S56, Y57, Y59, F60, P61, D62, Q65,Y102, D103 and D106 in SEQ ID NO: 8. As identified in FIG. 3, these arethe residues in the MuTFPI4F36 light and heavy chain sequences that havea heavy atom within a distance of 4 Å from a heavy atom when MuTFPI4F36is bound to the K2 domain of TFPI.

An antibody of the invention may comprise a CDR region from the specificantibody identified herein such as a CDR region from within SEQ ID NO: 4or 8. Such an antibody will preferably retain the ability to bind toTFPI as described herein. For example, as shown in FIG. 3, using theKabat definition, the CDR sequences within the light chain of MuTFPI4F36may be identified at amino acids 24 to 39, 55 to 61 and 94 to 102 of SEQID NO: 4 or SEQ ID NO: 6. The CDR sequences within the heavy chain ofMuTFPI4F36 may be identified at amino acids 31 to 35, 50 to 66 and 99 to110 of SEQ IS NO: 8 or SEQ ID NO: 10. An antibody of the invention maycomprise one or more of the CDR sequences shown in FIG. 3. For example,an antibody of the invention may comprise one, two or all three of theamino acid sequences shown at residues 24 to 39, 55 to 61 and 94 to 102of SEQ ID NO: 6. An antibody of the invention may alternatively oradditionally comprise one, two or all three of the amino acid sequencesshown at residues 31 to 35, 50 to 66 and 99 to 110 of SEQ ID NO: 10. Anantibody of the invention may comprise all six amino acid sequencesshown at residues 24 to 39, 55 to 61 and 94 to 102 of SEQ ID NO: 6 and31 to 35, 50 to 66 and 99 to 110 of SEQ ID NO: 10.

An antibody of the invention may be a humanized antibody, such as theantibody herein referred to as HzTFPI4F36 (mAbTFPI 2021). Such anantibody may comprise one or more CDR regions from within SEQ ID NO: 15or 18.

The heavy chain of an antibody according to the invention may comprise aCDR1 sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO: 18, whereinone of these amino acids may be substituted by a different amino acid.

The heavy chain of an antibody according to the invention may comprise aCDR2 sequence of amino acids 50 to 66 (TISRSGSYSYFPDSVQG) of SEQ ID NO:18, wherein one, two or three of these amino acids may be substituted bya different amino acid.

The heavy chain of an antibody according to the invention may comprise aCDR3 sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ ID NO: 18,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.

The light chain of an antibody according to the invention may comprise aCDR1 sequence of amino acids 24 to 39 (KSSQSLLESDGKTYLN) of SEQ ID NO:15, wherein one, two or three of these amino acids may be substitutedwith a different amino acid.

The light chain of an antibody according to the invention may comprise aCDR2 sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO: 15,wherein one or two of these amino acids may be substituted with adifferent amino acid.

The light chain of an antibody according to the invention may comprise aCDR3 sequence of amino acids 94 to 102 (LQATHFPQT) of SEQ ID NO: 15,wherein one or two of these amino acids may be substituted with adifferent amino acid.

More particularly, an antibody of the invention may have a heavy chainthat comprises:

-   -   a CDR1 sequence which, in turn, comprises amino acids 31 to 35        (NYAMS) of SEQ ID NO:18; and    -   a CDR2 sequence which, in turn, comprises amino acids 50 to 66        (TISRSGSYSYFPDSVQG) of SEQ ID NO:18; and    -   a CDR3 sequence which, in turn, comprises amino acids 99 to 110        (LGGYDEGDAMDS) of SEQ ID NO:18.

An antibody of the invention may have a light chain that comprises:

-   -   a CDR1 sequence which, in turn, comprises amino acids 24 to 39        (KSSQSLLESDGKTYLN) of SEQ ID NO: 15; and    -   a CDR2 sequence which, in turn, comprises amino acids 55 to 61        (LVSILDS) of SEQ ID NO: 15; and    -   a CDR3 sequence which, in turn, comprises amino acids 94 to 102        (LQATHFPQT) of SEQ ID NO: 15.

An antibody of the invention may comprise any combination of the aboveCDR regions.

More particularly, framework region 2 (FR2) of the heavy chain of anantibody of the invention may comprise amino acids:

-   -   T, in the position corresponding to position 40,    -   E, in the position corresponding to position 42,    -   R, in the position corresponding to position 44 and    -   A, in the position corresponding to position 49 of SEQ ID NO:        18.

Alternatively, said FR2 of the heavy chain may comprise amino acids 36to 49 of SEQ ID NO: 18.

An antibody of the invention may comprise any one of the following:

-   -   the VL amino acid sequence of SEQ ID NO: 15.    -   The VH amino acid sequence of SEQ ID NO: 18.    -   SEQ ID NOs: 15 and 18.    -   The light chain amino acid sequence of SEQ ID NO: 21.    -   The heavy chain amino acid sequence of SEQ ID NO: 24.    -   SEQ ID NOs: 21 and 24.

An antibody of the invention may alternatively be or may comprise avariant of one of these specific sequences such a variant of theMuTFPI4F36 antibody or a variant of HzTFPI4F36. For example, a variantmay be a substitution, deletion or addition variant of any of the aboveamino acid sequences.

A variant according to the current invention may be an antibody thatdoes not comprise:

-   -   N, in the position corresponding to position 31        of the CDR1 region of SEQ ID NO: 18;    -   R, in the position corresponding to position 53;    -   S, in the position corresponding to position 54;    -   S, in the position corresponding to position 56;    -   Y, in the position corresponding to position 57;    -   Y, in the position corresponding to position 59;    -   F, in the position corresponding to position 60;    -   P, in the position corresponding to position 61;    -   D, in the position corresponding to position 62; and    -   Q, in the position corresponding to position 65; of the CDR2        region of SEQ ID NO: 18.    -   Y, in the position corresponding to position 102;    -   D, in the position corresponding to position 103; and    -   D, in the position corresponding to position 106; of the CDR3        region of SEQ ID NO: 18.    -   E, in the position corresponding to position 31;    -   S, in the position corresponding to position 32;    -   D, in the position corresponding to position 33; and    -   Y, in the position corresponding to position 37; of the CDR1        region of SEQ ID NO: 15.    -   A, in the position corresponding to position 96;    -   T, in the position corresponding to position 97;    -   H, in the position corresponding to position 98; and    -   F, in the position corresponding to position 99; of the CDR3        region of SEQ ID NO: 15.

A variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to30 or more amino acid substitutions and/or deletions and/or insertionsfrom the specific sequences and fragments discussed above. “Deletion”variants may comprise the deletion of individual amino acids, deletionof small groups of amino acids such as 2, 3, 4 or 5 amino acids, ordeletion of larger amino acid regions, such as the deletion of specificamino acid domains or other features. “Insertion” variants may comprisethe insertion of individual amino acids, insertion of small groups ofamino acids such as 2, 3, 4 or 5 amino acids, or insertion of largeramino acid regions, such as the insertion of specific amino acid domainsor other features. “Substitution” variants preferably involve thereplacement of one or more amino acids with the same number of aminoacids and making conservative amino acid substitutions. For example, anamino acid may be substituted with an alternative amino acid havingsimilar properties, for example, another basic amino acid, anotheracidic amino acid, another neutral amino acid, another charged aminoacid, another hydrophilic amino acid, another hydrophobic amino acid,another polar amino acid, another aromatic amino acid or anotheraliphatic amino acid. Some properties of the 20 main amino acids whichcan be used to select suitable substituents are as follows:

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral Cys polar,hydrophobic, neutral Asn polar, hydrophilic, neutral Asp polar,hydrophilic, charged (−) Pro hydrophobic, neutral Glu polar,hydrophilic, charged (−) Gln polar, hydrophilic, neutral Phe aromatic,hydrophobic, neutral Arg polar, hydrophilic, charged (+) Gly aliphatic,neutral Ser polar, hydrophilic, neutral His aromatic, polar,hydrophilic, Thr polar, hydrophilic, neutral charged (+) Ile aliphatic,hydrophobic, neutral Val aliphatic, hydrophobic, neutral Lys polar,hydrophilic, charged (+) Trp aromatic, hydrophobic, neutral Leualiphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

Preferred “derivatives” or “variants” include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatized or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Substitutions may be, but are not limited to, conservativesubstitutions.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post-production modification, or whenthe antibody is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

In another aspect, the present invention features multispecificmolecules comprising an anti-TFPI antibody, or an antigen-fragmentthereof, of the invention. Such multispecific molecules includebispecific molecules comprising at least one first binding specificityfor TFPI and a second binding specificity for a second target epitope.One type of bispecific molecules are bispecific antibodies as known inthe art. Bispecific antibodies, or indeed multispcific antibodies, maybe prepared as full-length antibodies or antibody fragments (e.g.F(ab′)2 bispecific antibodies) or any other antigen-binding fragmentsdescribed herein.

In one aspect, the present invention features antibody derivatives (orimmunoconjugates), such as anti-TFPI antibodies conjugated or covalentlybound to a second agent. The second agent can be linked to the antibodydirectly or indirectiy, using any of a large number of available methodsknown to the person skilled in the art. For example, an agent can beattached at the hinge region of the reduced antibody component viadisulfide bond formation, using cross-linkers such as N-succinylS-(2-pyridyldithio) proprionate (SPDP), or via a carbohydrate moiety inthe Fc region of the antibody.

In one aspect, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, protein stability and/orantigen-dependent cellular cytotoxicity, or lack thereof. Furthermore,an antibody of the invention may be chemically modified (e.g., one ormore chemical moieties can be attached to the antibody) or be modifiedto alter its glycosylation, again to alter one or more functionalproperties of the antibody.

If desired, the class of an antibody may be “switched” by knowntechniques. For example, an antibody that was originally produced as anIgM molecule may be class switched to an IgG antibody. Class switchingtechniques also may be used to convert one IgG subclass to another, forexample: from IgG1 to IgG2 or IgG4; from IgG2 to IgG1 or IgG4; or fromIgG4 to IgG1 or IgG2. Engineering of antibodies to generate constantregion chimeric molecules, by combination of regions from different IgGsubclasses, can also be performed.

In one embodiment, the hinge region of CHI is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further for instancein U.S. Pat. No. 5,677,425 by Bodmer et al.

The constant region may further be modified to stabilize the antibody,e.g., to reduce the risk of a bivalent antibody separating into twomonovalent VH-VL fragments. For example, in an IgG4 constant region,residue 5241 may be mutated to a proline (P) residue to allow completedisulphide bridge formation at the hinge (see, e.g., Angal et al., MolImmunol. 199S; 30:105-8).

Variant antibodies according to the invention may have amino acidsequences which are more than 60%, or more than 65%, or more than 70%,or more than 75%, or more than 80%, preferably more than 85%, such asmore than 90%, such as more than 95% identical to SEQ ID NOs: 4 or 8, orfragments thereof. Other variant antibodies according to the inventionmay have amino acid sequences which are more than 60%, or more than 65%,or more than 70%, or more than 75%, or more than 80%, preferably morethan 85%, such as more than 90%, such as more than 95% identical to SEQID NOs: 15 or 18, or a fragment thereof. This level of amino acididentity may be seen across the full length of the relevant SEQ ID NOsequence or over a part of the sequence, such as across 20, 30, 40, 50,60, 70, 75, 80, 90, 100, 150, 200 or more amino acids, depending on thesize of the full length polypeptide.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, supra) with the following parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10,00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10,00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatized.

The present invention thus provides antibodies having specific VH and VLamino acid sequences and variants and fragments thereof which maintainthe function or activity of these VH and VL domains.

Accordingly, an antibody of the invention may comprise:

(a) a light chain variable region amino acid sequence of SEQ ID NO: 4;

(b) a fragment of at least 7 amino acids of (a) which retains theability to specifically bind to TFPI; or

(c) a variant of (a) having at least 70% amino acid sequence identity toa sequence of (a) and retaining the ability to specifically bind toTFPI.

An antibody of the invention may comprise:

(a) a heavy chain variable region amino acid sequence of SEQ ID NO: 8;

(b) a fragment of at least 7 amino acids of (a) which retains theability to specifically bind to TFPI; or

(c) a variant of (a) having at least 70% amino acid sequence identity toa sequence of (a) and retaining the ability to specifically bind toTFPI.

An antibody of the invention may comprise the light chain variableregion of SEQ ID NO: 4 and the heavy chain variable region of SEQ ID NO:8.

An antibody of the invention may comprise:

(a) the light chain variable region of SEQ ID NO: 4 and the heavy chainvariable region of SEQ ID NO: 8;

(b) a variant of (a) in which one or both of the heavy chain and lightchain sequences is modified such that it comprises a fragment of atleast 7 amino acids of the sequence specified in (a); or

(c) a variant of (a) or (b) in which one or both of the heavy and lightchain sequences is modified such that it has at least 70% amino acidsequence identity to a sequence of (a) or (b);

wherein the antibody retains the ability to specifically bind to TFPI.The antibody may also retain one or more additional functions oractivities of an antibody of the invention as described herein such asthe ability to inhibit TFPI or the ability to shorten clotting time,optionally without leading to a drop in platelet numbers.

Preferred fragments and variants of SEQ ID NO: 4 will comprise (i) aminoacids 24 to 39 of SEQ ID NO: 6; and/or (ii) amino acids 55 to 61 of SEQID NO: 6; and/or (iii) amino acids 94 to 102 of SEQ ID NO: 6. Preferredfragments and variants of SEQ ID NO: 8 will comprise (i) amino acids 31to 35 of SEQ ID NO: 10; and/or (ii) amino acids 50 to 66 of SEQ ID NO:10; and/or (iii) amino acids 99 to 110 of SEQ ID NO: 10.

Further preferred variants of SEQ ID NO: 4 will comprise amino acids 31to 33, 37 and 96 to 99 of SEQ ID NO: 6. Further preferred variants ofSEQ ID NO: 8 will comprise amino acids 31, 53, 54, 56, 57, 59, 60, 61,62, 65, 102, 103 and 106 of SEQ ID NO: 10.

An antibody of the invention may comprise:

(a) a light chain variable region amino acid sequence of SEQ ID NO: 15;

(b) a fragment of at least 7 amino acids of (a) which retains theability to specifically bind to TFPI; or

(c) a variant of (a) having at least 70% amino acid sequence identity toa sequence of (a) and retaining the ability to specifically bind toTFPI.

An antibody of the invention may comprise:

(a) a heavy chain variable region amino acid sequence of SEQ ID NO: 18;

(b) a fragment of at least 7 amino acids of (a) which retains theability to specifically bind to TFPI; or

(c) a variant of (a) having at least 70% amino acid sequence identity toa sequence of (a) and retaining the ability to specifically bind toTFPI.

An antibody of the invention may comprise the light chain variableregion of SEQ ID NO: 15 and the heavy chain variable region of SEQ IDNO: 18.

An antibody of the invention may comprise:

(a) the light chain variable region of SEQ ID NO: 15 and the heavy chainvariable region of SEQ ID NO: 18;

(b) a variant of (a) in which one or both of the heavy chain and lightchain sequences is modified such that it comprises a fragment of atleast 7 amino acids of the sequence specified in (a); or

(c) a variant of (a) or (b) in which one or both of the heavy and lightchain sequences is modified such that it has at least 70% amino acidsequence identity to a sequence of (a) or (b);

wherein the antibody retains the ability to specifically bind to TFPI.The antibody may also retain one or more additional functions oractivities of an antibody of the invention as described herein such asthe ability to inhibit TFPI or the ability to shorten clotting time,optionally without leading to a drop in platelet numbers.

As explained above, an antibody of the invention may bind to the sameepitope or region as another antibody of the invention. Thus it will beseen that such an antibody may bind to the same epitope or region ofTFPI as any of the specific antibodies, fragments and variants describedherein.

The invention also relates to polynucleotides that encode antibodies ofthe invention. Thus, a polynucleotide of the invention may encode anyantibody as described herein. The terms “nucleic acid molecule” and“polynucleotide” are used interchangeably herein and refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, messenger RNA (mRNA),cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA ofany sequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide of the invention may be provided in isolatedor purified form.

A nucleic acid sequence which “encodes” a selected polypeptide is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

In one embodiment, a polynucleotide of the invention comprises asequence which encodes a VH or VL amino acid sequence as describedabove. For example, a polynucleotide of the invention may encode apolypeptide comprising the sequence of SEQ ID NO: 4 or 8, or a variantor fragment thereof as described above. Such a polynucleotide mayconsist of or comprise a nucleic acid sequence of any one of SEQ ID NOs:3, 5, 7 and 9. A suitable polynucleotide sequence may alternatively be avariant of one of these specific polynucleotide sequences. For example,a variant may be a substitution, deletion or addition variant of any ofthe above nucleic acid sequences. A variant polynucleotide may comprise1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to75 or more nucleic acid substitutions and/or deletions from thesequences given in the sequence listing.

Suitable variants may be at least 70% homologous to a polynucleotide ofany one of SEQ ID NOs: 3, 5, 7 and 9 preferably at least 80 or 90% andmore preferably at least 95%, 97% or 99% homologous thereto. Methods ofmeasuring homology are well known in the art and it will be understoodby those of skill in the art that in the present context, homology iscalculated on the basis of nucleic acid identity. Such homology mayexist over a region of at least 15, preferably at least 30, for instanceat least 40, 60, 100, 200 or more contiguous nucleotides. Such homologymay exist over the entire length of the unmodified polynucleotidesequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example, the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al (1984) Nucleic Acids Research 12, p 387-395).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S,F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4,and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

The homologue may differ from a sequence in the relevant polynucleotideby less than 3, 5, 10, 15, 20 or more mutations (each of which may be asubstitution, deletion or insertion). These mutations may be measuredover a region of at least 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specificsequences given in the sequence listing by virtue of the redundancy inthe genetic code. The DNA code has 4 primary nucleic acid residues (A,T, C and G) and uses these to “spell” three letter codons whichrepresent the amino acids the proteins encoded in an organism's genes.The linear sequence of codons along the DNA molecule is translated intothe linear sequence of amino acids in the protein(s) encoded by thosegenes. The code is highly degenerate, with 61 codons coding for the 20natural amino acids and 3 codons representing “stop” signals. Thus, mostamino acids are coded for by more than one codon—in fact several arecoded for by four or more different codons. A variant polynucleotide ofthe invention may therefore encode the same polypeptide sequence asanother polynucleotide of the invention, but may have a differentnucleic acid sequence due to the use of different codons to encode thesame amino acids.

Polynucleotide “fragments” according to the invention may be made bytruncation, e.g. by removal of one or more nucleotides from one or bothends of a polynucleotide. Up to 10, up to 20, up to 30, up to 40, up to50, up to 75, up to 100, up to 200 or more amino acids may be removedfrom the 3′ and/or 5′ end of the polynucleotide in this way. Fragmentsmay also be generated by one or more internal deletions. Such fragmentsmay be derived from a sequence of SEQ ID NOs: 3, 5, 7 and 9 or may bederived from a variant polynucleotide as described herein. Preferablysuch fragments are between 30 and 300 residues in length, e.g. 30 to300, 30 to 200, 30 to 100, 100 to 200 or 200 to 300 residues.Alternatively, fragments of the invention may be longer sequences, forexample comprising at least 50%, at least 60%, at least 70%, at least80% or at least 90% of a full length polynucleotide of the invention.

An antibody of the invention may thus be produced from or delivered inthe form of a polynucleotide which encodes, and is capable ofexpressing, it. Where the antibody comprises two or more chains, apolynucleotide of the invention may encode one or more antibody chains.For example, a polynucleotide of the invention may encode an antibodylight chain, an antibody heavy chain or both. Two polynucleotides may beprovided, one of which encodes an antibody light chain and the other ofwhich encodes the corresponding antibody heavy chain. Such apolynucleotide or pair of polynucleotides may be expressed together suchthat an antibody of the invention is generated.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Sambrook et al(1989, Molecular Cloning—a laboratory manual; Cold Spring Harbor Press).

The nucleic acid molecules of the present invention may be provided inthe form of an expression cassette which includes control sequences,signal peptide sequences operably linked to the inserted sequence, thusallowing for expression of the antibody of the invention in vivo. Theseexpression cassettes, in turn, are typically provided within vectors(e.g., plasmids or recombinant viral vectors). Such an expressioncassette may be administered directly to a host subject. Alternatively,a vector comprising a polynucleotide of the invention may beadministered to a host subject. Preferably the polynucleotide isprepared and/or administered using a genetic vector. A suitable vectormay be any vector which is capable of carrying a sufficient amount ofgenetic information, and allowing expression of a polypeptide of theinvention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancers,signal peptide sequences and other elements, such as for examplepolyadenylation signals which may be necessary, and which are positionedin the correct orientation, in order to allow for expression of apeptide of the invention. Other suitable vectors would be apparent topersons skilled in the art. By way of further example in this regard werefer to Sambrook et al.

The invention also includes cells that have been modified to express anantibody of the invention. Such cells include transient, or preferablystable higher eukaryotic cell lines, such as mammalian cells or insectcells, lower eukaryotic cells, such as yeast or prokaryotic cells suchas bacterial cells. Particular examples of cells which may be modifiedby insertion of vectors or expression cassettes encoding for an antibodyof the invention include mammalian HEK293, CHO, BHK, NSO and humanretina cells. Preferably the cell line selected will be one which is notonly stable, but also allows for mature glycosylation and cell surfaceexpression of a polypeptide.

Such cell lines of the invention may be cultured using routine methodsto produce an antibody of the invention, or may be used therapeuticallyor prophylactically to deliver antibodies of the invention to a subject.Alternatively, polynucleotides, expression cassettes or vectors of theinvention may be administered to a cell from a subject ex vivo and thecell then returned to the body of the subject.

In another aspect, the present invention provides compositions andformulations comprising molecules of the invention, such as theantibodies, polynucleotides, vectors and cells described herein. Forexample, the invention provides a pharmaceutical composition comprisingone or more molecules of the invention, such as one or more antibodiesof the invention, formulated together with a pharmaceutically acceptablecarrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forparenteral, e.g. intravenous, intramuscular or subcutaneousadministration (e.g., by injection or infusion). Depending on the routeof administration, the antibody may be coated in a material to protectthe antibody from the action of acids and other natural conditions thatmay inactivate or denature the antibody.

Preferred pharmaceutically acceptable carriers comprise aqueous carriersor diluents. Examples of suitable aqueous carriers that may be employedin the pharmaceutical compositions of the invention include water,buffered water and saline. Examples of other carriers include ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition.

A pharmaceutical composition of the invention also may include apharmaceutically acceptable anti-oxidant. These compositions may alsocontain adjuvants such as preservatives, wetting agents, emulsifyingagents and dispersing agents. Prevention of presence of microorganismsmay be ensured both by sterilization procedures, supra, and by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration.

Sterile injectable solutions can be prepared by incorporating the activeagent (e.g. antibody) in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the active agent into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active agent plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Pharmaceutical compositions of the invention may comprise additionalactive ingredients as well as an antibody of the invention. As mentionedabove, compositions of the invention may comprise one or more antibodiesof the invention. They may also comprise additional therapeutic orprophylactic agents. For example, where a pharmaceutical composition ofthe invention is intended for use in the treatment of a bleedingdisorder, it may additionally comprise one or more agents intended toreduce the symptoms of the bleeding disorder. For example, thecomposition may comprise one or more clotting factors. The compositionmay comprise one or more other components intended to improve thecondition of the patient. For example, where the composition is intendedfor use in the treatment of patients suffering from unwanted bleedingsuch as patients undergoing surgery or patients suffering from trauma,the composition may comprise one or more analgesic, anaesthetic,immunosuppressant or anti-inflammatory agents. Also falling within thescope of the present invention are kits comprising antibodies or othercompositions of the invention and instructions for use. Such a kit mayfurther contain one ore more additional reagents, such as an additionaltherapeutic or prophylactic agent as discussed above.

The antibodies, other molecules and compositions of the presentinvention have numerous in vitro and in vivo therapeutic utilitiesinvolving the treatment and prevention of clotting related disorders.For example, these antibodies and compositions can be administered tocells in culture, in vitro or ex vivo, or to human subjects, e.g., invivo, to prevent or treat a variety of disorders.

In particular, the present invention provides methods for the treatmentof bleeding disorders or for the enhancement of blood clottingcomprising administering to a patient in need thereof an effectiveamount of an antibody or other molecule or composition of the invention.For example, such methods may be for the treatment of clotting factordeficiencies such as haemophilia A, haemophilia B, Factor XI deficiency,Factor VII deficiency, thrombocytopenia or von Willebrand's disease.Such methods may be for the treatment of conditions accompanied by thepresence of a clotting factor inhibitor. Such methods may be for thetreatment of excessive bleeding. The antibodies and compositions of theinvention may be used to treat patients before, during, or after surgeryor anticoagulant therapy or after trauma. The antibodies andcompositions described herein may be used in any such treatment or maybe used in the manufacture of a medicament for use in any suchtreatment.

The antibodies and compositions of the present invention may beadministered for prophylactic/preventitive and/or therapeutictreatments.

In therapeutic applications, antibodies or compositions are administeredto a subject already suffering from a disorder or condition as describedabove, in an amount sufficient to cure, alleviate or partially arrestthe condition or one or more of its symptoms. Such therapeutic treatmentmay result in a decrease in severity of disease symptoms, or an increasein frequency or duration of symptom-free periods. An amount adequate toaccomplish this is defined as“therapeutically effective amount”. Forexample, where the treatment is for unwanted bleeding, therapy may bedefined as a decrease in the amount of bleeding or suitable coagulationto stop the bleeding altogether.

In prophylactic or preventitive applications, antibodies or compositionsare administered to a subject at risk of a disorder or condition asdescribed above, in an amount sufficient to prevent or reduce thesubsequent effects of the condition or one or more of its symptoms. Anamount adequate to accomplish this is defined as a “prophylacticallyeffective amount”. For example, where the treatment is to preventunwanted bleeding, a prophylactic effect may be defined as theprevention of bleeding or a reduced period or quantity of bleedingcompared to that that would be seen in the absence of the modulator.

Effective amounts for each purpose will depend on the severity of thedisease or injury as well as the weight and general state of thesubject.

As used herein, the term “subject” includes any human or non-humananimal. The term “non-human animal” includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

An antibody or composition of the present invention may be administeredvia one or more routes of administration using one or more of a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. Preferred routes of administration forantibodies or compositions of the invention include intravenous,intramuscular, intradermal, intraperitoneal, subcutaneous, spinal orother parenteral routes of administration, for example by injection orinfusion. The phrase “parenteral administration” as used herein meansmodes of administration other than enteral and topical administration,usually by injection. Alternatively, an antibody or composition of theinvention can be administered via a non-parenteral route, such as atopical, epidermal or mucosal route of administration.

Similarly, an antibody of the invention may be used for the manufactureof a medicament suitable for parenteral administration.

An antibody of the invention may be used for the manufacture of amedicament suitable for intravenous administration.

An antibody of the invention may be used for the manufacture of amedicament suitable for intramuscular administration.

An antibody of the invention may be used for the manufacture of amedicament suitable for subcutaneous administration.

A suitable dosage of an antibody of the invention may be determined by askilled medical practitioner. Actual dosage levels of the activeingredients in the pharmaceutical compositions of the present inventionmay be varied so as to obtain an amount of the active ingredient whichis effective to achieve the desired therapeutic response for aparticular patient, composition, and mode of administration, withoutbeing toxic to the patient. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular antibody employed, the route of administration, the time ofadministration, the rate of excretion of the antibody, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A suitable dose of an antibody of the invention may be, for example, inthe range of from about 0.1 μg/kg to about 100 mg/kg body weight of thepatient to be treated. For example, a suitable dosage may be from about1 μg/kg to about 10 mg/kg body weight per day or from about 1 mg/kg toabout 5 mg/kg body weight per day. A suitable dose of an antibody of theinvention may be in the range of from 2 to 200 mg/kg, such as about150-200 mg/kg, such as about 150-170 mg/kg, such as about 100-150 mg/kg,such as about 50-100 mg/kg, such as about 70-90 mg/kg, such as about10-50 mg/kg, such as about 10-30 mg/kg.

Other suitable dosages may be approximately 0.1-10 mg/kg, such asapproximately 0.1-1 mg/kg, such as approximately 1-2 mg/kg orapproximately 2-3 mg/kg or approximately 4-5 mg/kg or approximately 5-6mg/kg or approximately 6-7 mg/kg or approximately 7-8 mg/kg orapproximately 8-9 mg/kg or approximately 9-10 mg/kg; or approximately10-21 mg/kg, such as approximately 10-11 mg/kg, or approximately 11-12mg/kg, or approximately 12-13 mg/kg, or approximately 13-14 mg/kg, orapproximately 14-15 mg/kg, or approximately 15-16 mg/kg, orapproximately 16-17 mg/kg, or approximately 17-18 mg/kg, orapproximately 18-19 mg/kg, or approximately 19-20 mg/kg or approximately20-21 mg/kg.

The amount of monoclonal antibody administered to a subject may be suchthat its administration results in a subject plasma concentration ofabout 10 μg/ml to about 40 μg/ml, such as about 15-35 μg/ml, such asabout 10-15 μg/ml, such as about 15-20 μg/ml, such as about 20-25 μg/ml,such as about 25-30 μg/ml, such as about 30-35 μg/ml, such as about35-40 μg/ml, of said monoclonal antibody. Dosage regimens may beadjusted to provide the optimum desired response (e.g., a therapeuticresponse). For example, a single bolus may be administered, severaldivided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Antibodies may be administered in a single dose or in multiple doses.The multiple doses may be administered via the same or different routesand to the same or different locations. Alternatively, antibodies can beadministered as a sustained release formulation, in which case lessfrequent administration is required. Dosage and frequency may varydepending on the half-life of the antibody in the patient and theduration of treatment that is desired. The dosage and frequency ofadministration can also vary depending on whether the treatment isprophylactic or therapeutic. In prophylactic applications, a relativelylow dosage may be administered at relatively infrequent intervals over along period of time. In therapeutic applications, a relatively highdosage may be administered, for example until the patient shows partialor complete amelioration of symptoms of disease.

Thus, an antibody of the invention may be administered: approximatelydaily, approximately every other day, approximately every third day,approximately every fourth day, approximately every fifth day,approximately every sixth day; approximately every week, such as every5, 6, 7, 8, 9 or 10 days; approximately every other week, such as every11, 12, 13, 14, 15, 16 or 17 days; approximately every third week, suchas every 18, 19, 20, 21, 22, 23 or 24 days; approximately every fourthweek, such as every 25, 26, 27, 28, 29, 30 or 31 days. An antibody ofthe invention may also be administered on-demand.

As mentioned above, antibodies of the invention may be co-administeredwith one or other more other therapeutic agents. The other agent may bean agent that will enhance the effects of the modulator. The other agentmay be an agent that acts to enhance blood coagulation, such as a bloodcoagulation factor. In particular, the modulators of the invention maybe co-administered with Factor VII(a) or FVIII(a). The other agent maybe intended to treat other symptoms or conditions of the patient. Forexample, the other agent may be an analgesic, anaesthetic,immunosuppressant or anti-inflammatory agent.

Combined administration of two or more agents may be achieved in anumber of different ways. In one embodiment, the antibody and the otheragent may be administered together in a single composition. In anotherembodiment, the antibody and the other agent may be administered inseparate compositions as part of a combined therapy. For example, themodulator may be administered before, after or concurrently with theother agent.

The term “treatment”, as used herein, refers to the medical therapy ofany human or other animal subject in need thereof. Said subject isexpected to have undergone physical examination by a medicalpractitioner or a veterinary medical practitioner, who has given atentative or definitive diagnosis which would indicate that the use ofsaid specific treatment is beneficial to the health of said human orother animal subject. The timing and purpose of said treatment may varyfrom one individual to another, according to the status quo of thesubject's health. Thus, said treatment may be prophylactic, palliative,symptomatic and/or curative. In terms of the present invention,prophylactic, palliative, symptomatic and/or curative treatments mayrepresent separate aspects of the invention.

Thus, an antibody of the invention may be administered parenterally.

An antibody of the invention may be administered intravenously.

An antibody of the invention may be administered intramuscularly.

An antibody of the invention may be administered subcutaneously.

An antibody of the invention may be administered prophylactically,

An antibody of the invention may be administered therapeutically (ondemand).

An antibody of the invention may be capable of significantly reducingblood loss.

An antibody of the invention may be capable of significantly reducingbleeding time.

Thus, the invention is also a method of treating a subject in needthereof with a monoclonal antibody that is capable of binding the K2domain of TFPI, wherein the amount of monoclonal antibody administeredis such as to saturate its target. The amount of monoclonal antibodyadministered may be such as to saturate soluble TFPI. The amount ofmonoclonal antibody administered may be such as to saturateendothelium-bound TFPI.

The term “coagulopathy”, as used herein, refers to an increasedhaemorrhagic tendency which may be caused by any qualitative orquantitative deficiency of any pro-coagulative component of the normalcoagulation cascade, or any upregulation of fibrinolysis. Suchcoagulopathies may be congenital and/or acquired and/or iatrogenic andare identified by a person skilled in the art.

Non-limiting examples of congenital hypocoagulopathies are haemophiliaA, haemophilia B, Factor VII deficiency, Factor XI deficiency, vonWillebrand's disease and thrombocytopenias such as Glanzmann'sthombasthenia and Bernard-Soulier syndrome.

A non-limiting example of an acquired coagulopathy is serine proteasedeficiency caused by vitamin K deficiency; such vitamin K-deficiency maybe caused by administration of a vitamin K antagonist, such as warfarin.Acquired coagulopathy may also occur following extensive trauma. In thiscase otherwise known as the “bloody vicious cycle”, it is characterisedby haemodilution (dilutional thrombocytopaenia and dilution of clottingfactors), hypothermia, consumption of clotting factors and metabolicderangements (acidosis). Fluid therapy and increased fibrinolysis mayexaserbate this situation. Said haemorrhage may be from any part of thebody.

Haemophilia A with “inhibitors” (that is, allo-antibodies against factorVIII) and haemophilia B with “inhibitors” (that is, allo-antibodiesagainst factor IX) are non-limiting examples of coagulopathies that arepartly congenital and partly acquired.

A non-limiting example of an iatrogenic coagulopathy is an overdosage ofanticoagulant medication—such as heparin, aspirin, warfarin and otherplatelet aggregation inhibitors—that may be prescribed to treatthromboembolic disease. A second, non-limiting example of iatrogeniccoagulopathy is that which is induced by excessive and/or inappropriatefluid therapy, such as that which may be induced by a blood transfusion.

In one embodiment of the current invention, haemorrhage is associatedwith haemophilia A or B. In another embodiment, haemorrhage isassociated with haemophilia A or B with acquired inhibitors. In anotherembodiment, haemorrhage is associated with thrombocytopenia. In anotherembodiment, haemorrhage is associated with von Willebrand's disease. Inanother embodiment, haemorrhage is associated with severe tissue damage.In another embodiment, haemorrhage is associated with severe trauma. Inanother embodiment, haemorrhage is associated with surgery. In anotherembodiment, haemorrhage is associated with haemorrhagic gastritis and/orenteritis. In another embodiment, the haemorrhage is profuse uterinebleeding, such as in placental abruption. In another embodiment,haemorrhage occurs in organs with a limited possibility for mechanicalhaemostasis, such as intracranially, intraaurally or intraocularly. Inanother embodiment, haemorrhage is associated with anticoagulanttherapy.

An antibody of the current invention may be used to treat a subject witha coagulopathy. Thus, the invention is also the use of a monoclonalantibody, that is capable of binding the K2 domain of TFPI, for thetreatment of a subject in need thereof; as well as use of said antibodyfor the manufacture of a medicament for the treatment of a subject inneed thereof. Furthermore, the invention is a method of treating asubject in need thereof with a monoclonal antibody that is capable ofbinding to the K2 domain of TFPI.

Use of said monoclonal antibody of the invention may significantlyreduce blood loss.

Use of said monoclonal antibody of the invention may significantlyreduce bleeding time.

Furthermore, use of said monoclonal antibody of the invention may reducein vivo clotting time without causing transient thrombocytopaenia.

EMBODIMENTS

The following is a non-limiting list of embodiments of the presentinvention:

Embodiment 1

A monoclonal antibody that is capable of specifically binding the K2domain of TFPI, wherein said antibody is capable of binding an epitopecomprising one or more residues selected from the group consisting ofE10, E11, D12, P13, R17, Y19, T21, Y23, F24, N26, Q28, Q31, C32, E33,R34, F35, K36 and L50 of SEQ ID NO: 2.

Embodiment 2

The monoclonal antibody according to embodiment 1, wherein said antibodyis capable of specifically binding an epitope comprising an epitopecomprising residue E10 of SEQ ID NO: 2.

Embodiment 3

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising an epitope comprising residue E11 of SEQ ID NO: 2.

Embodiment 4

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue D12 of SEQ ID NO: 2.

Embodiment 5

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue P13 of SEQ ID NO: 2.

Embodiment 6

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue R17 of SEQ ID NO: 2.

Embodiment 7

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue Y19 of SEQ ID NO: 2.

Embodiment 8

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue T21 of SEQ ID NO: 2.

Embodiment 9

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue Y23 of SEQ ID NO: 2.

Embodiment 10

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue F24 of SEQ ID NO: 2.

Embodiment 11

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue N26 of SEQ ID NO: 2.

Embodiment 12

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue Q28 of SEQ ID NO: 2.

Embodiment 13

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue Q31 of SEQ ID NO: 2.

Embodiment 14

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue C32 of SEQ ID NO: 2.

Embodiment 15

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue E33 of SEQ ID NO: 2.

Embodiment 16

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue R34 of SEQ ID NO: 2.

Embodiment 17

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue F35 of SEQ ID NO: 2.

Embodiment 18

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue K36 of SEQ ID NO: 2.

Embodiment 19

The monoclonal antibody according to any one of the above embodiments,wherein said antibody is capable of specifically binding an epitopecomprising residue L50 of SEQ ID NO: 2.

Embodiment 20

The monoclonal antibody according to any one of embodiments 1-16 and18-19, wherein said antibody is capable of specifically binding anepitope comprising residues E10, E11, D12, P13, R17, Y19, T21, Y23, F24,N26, Q28, Q31, C32, E33, R34, K36 and L50 of SEQ ID NO: 2.

Embodiment 21

The monoclonal antibody according to any one of embodiments 1-3, 5-9,12-13 and 15-19, wherein said antibody is capable of specificallybinding an epitope comprising residues E10, E11, P13, R17, Y19, T21,Y23, Q28, Q31, E33, R34, F35, K36 and L50 of SEQ ID NO: 2.

Embodiment 22

A monoclonal antibody that is capable of binding the K2 domain of TFPI,wherein the light chain of said antibody comprises amino acid residues:

-   -   E, in the position corresponding to position 31,    -   S, in the position corresponding to position 32,    -   D, in the position corresponding to position 33,    -   Y, in the position corresponding to position 37,    -   A, in the position corresponding to position 96,    -   T, in the position corresponding to position 97 and    -   F, in the position corresponding to position 99 of SEQ ID NO:        15;        and wherein the heavy chain of said antibody comprises amino        acid residues:    -   N, in the position corresponding to position 31,    -   R, in the position corresponding to position 53,    -   S, in the position corresponding to position 54,    -   Y, in the position corresponding to position 57,    -   Y, in the position corresponding to position 59,    -   F, in the position corresponding to position 60,    -   P, in the position corresponding to position 61,    -   D, in the position corresponding to position 62,    -   Q, in the position corresponding to position 65,    -   Y, in the position corresponding to position 102,    -   D, in the position corresponding to position 103 and    -   D, in the position corresponding to position 106 of SEQ ID NO:        18.

Embodiment 23

A monoclonal antibody according to any of claims 1-21, wherein the lightchain of said antibody comprises amino acid residues:

-   -   E, in the position corresponding to position 31,    -   S, in the position corresponding to position 32,    -   D, in the position corresponding to position 33,    -   Y, in the position corresponding to position 37,    -   A, in the position corresponding to position 96,    -   T, in the position corresponding to position 97 and    -   F, in the position corresponding to position 99 of SEQ ID NO:        15;        and wherein the heavy chain of said antibody comprises amino        acid residues:    -   N, in the position corresponding to position 31,    -   R, in the position corresponding to position 53,    -   S, in the position corresponding to position 54,    -   Y, in the position corresponding to position 57,    -   Y, in the position corresponding to position 59,    -   F, in the position corresponding to position 60,    -   P, in the position corresponding to position 61,    -   D, in the position corresponding to position 62,    -   Q, in the position corresponding to position 65,    -   Y, in the position corresponding to position 102,    -   D, in the position corresponding to position 103 and    -   D, in the position corresponding to position 106 of SEQ ID NO:        18.

Embodiment 24

The monoclonal antibody according to embodiment 22 or embodiment 23,wherein said heavy chain further comprises an S in the positioncorresponding to position 52 of SEQ ID NO: 18.

Embodiment 25

The monoclonal antibody according to any one of embodiments 22-23,wherein said light chain further comprises an H in the positioncorresponding to position 98 of SEQ ID NO: 15 and said heavy chainfurther comprises an S in the position corresponding to position 56 ofSEQ ID NO: 18.

Embodiment 26

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the heavychain of said antibody comprises a CDR1 sequence of amino acids 31 to 35(NYAMS) of SEQ ID NO:18, wherein one of these amino acids may besubstituted by a different amino acid.

Embodiment 27

A monoclonal antibody according to any of claims 1 to 21 that is capableof binding the Kunitz 2 (K2) domain of tissue factor pathway inhibitor(TFPI), wherein the heavy chain of said antibody comprises a CDR1sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO:18, wherein one ofthese amino acids may be substituted by a different amino acid.

Embodiment 28

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the heavychain of said antibody comprises a CDR2 sequence of amino acids 50 to 66(TISRSGSYSYFPDSVQG) of SEQ ID NO: 18, wherein one, two or three of theseamino acids may be substituted by a different amino acid.

Embodiment 29

A monoclonal antibody according to any of claims 1 to 21 that is capableof binding the Kunitz 2 (K2) domain of tissue factor pathway inhibitor(TFPI),

wherein the heavy chain of said antibody comprises a CDR2 sequence ofamino acids 50 to 66 (TISRSGSYSYFPDSVQG) of SEQ ID NO: 18, wherein one,two or three of these amino acids may be substituted by a differentamino acid.

Embodiment 30

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the heavychain of said antibody comprises a CDR3 sequence of amino acids 99 to110 (LGGYDEGDAMDS) of SEQ ID NO: 18, wherein one, two or three of theseamino acids may be substituted by a different amino acid.

Embodiment 31

A monoclonal antibody according to any of claims 1 to 21 that is capableof binding the Kunitz 2 (K2) domain of tissue factor pathway inhibitor(TFPI), wherein the heavy chain of said antibody comprises a CDR3sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ ID NO: 18,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.

Embodiment 32

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the lightchain of said antibody comprises a CDR1 sequence of amino acids 24 to 39(KSSQSLLESDGKTYLN) of SEQ ID NO: 15, wherein one, two or three of theseamino acids may be substituted with a different amino acid.

Embodiment 33

A monoclonal antibody according to any of claims 1 to 21 that is capableof binding the Kunitz 2 (K2) domain of tissue factor pathway inhibitor(TFPI), wherein the light chain of said antibody comprises a CDR1sequence of amino acids 24 to 39 (KSSQSLLESDGKTYLN) of SEQ ID NO: 15,wherein one, two or three of these amino acids may be substituted with adifferent amino acid.

Embodiment 34

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the lightchain of said antibody comprises a CDR2 sequence of amino acids 55 to 61(LVSILDS) of SEQ ID NO: 15, wherein one or two of these amino acids maybe substituted with a different amino acid.

Embodiment 35

A monoclonal antibody according to any of claims 1 to 21 that is capableof binding the Kunitz 2 (K2) domain of tissue factor pathway inhibitor(TFPI), wherein the light chain of said antibody comprises a CDR2sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO: 15, wherein oneor two of these amino acids may be substituted with a different aminoacid.

Embodiment 36

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the lightchain of said antibody comprises a CDR3 sequence of amino acids 94 to102 (LQATHFPQT) of SEQ ID NO: 15, wherein one or two of these aminoacids may be substituted with a different amino acid.

Embodiment 37

A monoclonal antibody according to any of claims 1 to 21 that is capableof binding the Kunitz 2 (K2) domain of tissue factor pathway inhibitor(TFPI), wherein the light chain of said antibody comprises a CDR3sequence of amino acids 94 to 102 (LQATHFPQT) of SEQ ID NO: 15, whereinone or two of these amino acids may be substituted with a differentamino acid.

Embodiment 38

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the heavychain of said antibody comprises:

-   -   a CDR1 sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO:18,        wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 50 to 66 (TISRSGSYSYFPDSVQG) of        SEQ ID NO:18, wherein one, two or three of these amino acids may        be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ        ID NO:18, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 39

A monoclonal antibody according to any of claims 1 to 21, wherein theheavy chain of said antibody comprises:

-   -   a CDR1 sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO:18,        wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 50 to 66 (TISRSGSYSYFPDSVQG) of        SEQ ID NO:18, wherein one, two or three of these amino acids may        be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ        ID NO:18, wherein one, two or three of these amino acids may be        substituted by a different amino acid.

Embodiment 40

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the lightchain of said antibody comprises:

-   -   a CDR1 sequence of amino acids 24 to 39 (KSSQSLLESDGKTYLN) of        SEQ ID NO: 15, wherein one, two or three of these amino acids        may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO:        15, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 94 to 102 (LQATHFPQT) of SEQ ID        NO: 15, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 41

A monoclonal antibody according to any of claims 1 to 21, wherein thelight chain of said antibody comprises:

-   -   a CDR1 sequence of amino acids 24 to 39 (KSSQSLLESDGKTYLN) of        SEQ ID NO: 15, wherein one, two or three of these amino acids        may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO:        15, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 94 to 102 (LQATHFPQT) of SEQ ID        NO: 15, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 42

A monoclonal antibody that is capable of binding the Kunitz 2 (K2)domain of tissue factor pathway inhibitor (TFPI), wherein the heavychain of said antibody comprises:

-   -   a CDR1 sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO:18,        wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 50 to 66 (TISRSGSYSYFPDSVQG) of        SEQ ID NO:18, wherein one, two or three of these amino acids may        be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ        ID NO:18, wherein one, two or three of these amino acids may be        substituted by a different amino acid;        and wherein the light chain of said antibody comprises:    -   a CDR1 sequence of amino acids 24 to 39 (KSSQSLLESDGKTYLN) of        SEQ ID NO: 15, wherein one, two or three of these amino acids        may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO:        15, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 94 to 102 (LQATHFPQT) of SEQ ID        NO: 15, wherein one or two of these amino acids may be        substituted with a different amino acid.

Embodiment 43

A monoclonal antibody according to any of claims 1 to 21, wherein theheavy chain of said antibody comprises:

-   -   a CDR1 sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO:18,        wherein one of these amino acids may be substituted by a        different amino acid; and/or    -   a CDR2 sequence of amino acids 50 to 66 (TISRSGSYSYFPDSVQG) of        SEQ ID NO:18, wherein one, two or three of these amino acids may        be substituted by a different amino acid; and/or    -   a CDR3 sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ        ID NO:18, wherein one, two or three of these amino acids may be        substituted by a different amino acid;        and wherein the light chain of said antibody comprises:    -   a CDR1 sequence of amino acids 24 to 39 (KSSQSLLESDGKTYLN) of        SEQ ID NO: 15, wherein one, two or three of these amino acids        may be substituted with a different amino acid; and/or    -   a CDR2 sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO:        15, wherein one or two of these amino acids may be substituted        with a different amino acid; and/or    -   a CDR3 sequence of amino acids 94 to 102 (LQATHFPQT) of SEQ ID        NO: 15,        wherein one or two of these amino acids may be substituted with        a different amino acid.

Embodiment 44

A monoclonal antibody according to any one of embodiments 26-43, whereinsaid amino acid substitutions do not comprise amino acids:

-   -   N, in the position corresponding to position 31 of the CDR1        region of SEQ ID NO: 18;    -   R, in the position corresponding to position 53;    -   S, in the position corresponding to position 54;    -   S, in the position corresponding to position 56;    -   Y, in the position corresponding to position 57;    -   Y, in the position corresponding to position 59;    -   F, in the position corresponding to position 60;    -   P, in the position corresponding to position 61;    -   D, in the position corresponding to position 62; and    -   Q, in the position corresponding to position 65;        of the CDR2 region of SEQ ID NO: 18.    -   Y, in the position corresponding to position 102;    -   D, in the position corresponding to position 103; and    -   D, in the position corresponding to position 106;        of the CDR3 region of SEQ ID NO: 18.    -   E, in the position corresponding to position 31;    -   S, in the position corresponding to position 32;    -   D, in the position corresponding to position 33; and    -   Y, in the position corresponding to position 37;        of the CDR1 region of SEQ ID NO: 15.    -   A, in the position corresponding to position 96;    -   T, in the position corresponding to position 97;    -   H, in the position corresponding to position 98; and    -   F, in the position corresponding to position 99;        of the CDR3 region of SEQ ID NO: 15.

Embodiment 45

The monoclonal antibody according to any one of embodiments 26-44,wherein said amino acid substitution is a conservative substitution.

Embodiment 46

The monoclonal antibody according to any one of embodiments 26-45,wherein the heavy chain of said antibody comprises:

-   -   a CDR1 sequence that comprises amino acids 31 to 35 (NYAMS) of        SEQ ID NO:18; and    -   a CDR2 sequence that comprises amino acids 50 to 66        (TISRSGSYSYFPDSVQG) of SEQ ID NO:18; and    -   a CDR3 sequence that comprises amino acids 99 to 110        (LGGYDEGDAMDS) of SEQ ID NO:18.

Embodiment 47

The monoclonal antibody according to any one of embodiments 26-46,wherein the light chain of said antibody comprises:

-   -   a CDR1 sequence that comprises amino acids 24 to 39        (KSSQSLLESDGKTYLN) of SEQ ID NO: 15; and    -   a CDR2 sequence that comprises amino acids 55 to 61 (LVSILDS) of        SEQ ID NO: 15; and    -   a CDR3 sequence that comprises amino acids 94 to 102 (LQATHFPQT)        of SEQ ID NO: 15.

Embodiment 48

The monoclonal antibody according to any one of embodiments 46-47,wherein the heavy chain comprises:

-   -   a CDR1 sequence that comprises amino acids 31 to 35 (NYAMS) of        SEQ ID NO:18; and    -   a CDR2 sequence that comprises amino acids 50 to 66        (TISRSGSYSYFPDSVQG) of SEQ ID NO:18; and    -   a CDR3 sequence that comprises amino acids 99 to 110        (LGGYDEGDAMDS) of SEQ ID NO:18;        and wherein the light chain comprises:    -   a CDR1 sequence that comprises amino acids 24 to 39        (KSSQSLLESDGKTYLN) of SEQ ID NO: 15; and    -   a CDR2 sequence that comprises amino acids 55 to 61 (LVSILDS) of        SEQ ID NO: 15; and    -   a CDR3 sequence that comprises amino acids 94 to 102 (LQATHFPQT)        of SEQ ID NO: 15.

Embodiment 49

The monoclonal antibody according to any one of the precedingembodiments, wherein the light chain of said antibody comprises SEQ IDNO: 15.

Embodiment 50

The monoclonal antibody according to any one of the precedingembodiments, wherein the heavy chain of said antibody comprises SEQ IDNO: 18.

Embodiment 51

The monoclonal antibody according to any one of the preceedingembodiments, wherein said antibody comprises SEQ ID NO: 15 and SEQ IDNO: 18.

Embodiment 52

The monoclonal antibody according to any one of the preceedingembodiments, wherein said antibody comprises the light chain of SEQ IDNO: 21

Embodiment 53

The monoclonal antibody according to any one of the preceedingembodiments, wherein said antibody comprises the heavy chain of SEQ IDNO: 24.

Embodiment 54

The monoclonal antibody according to any one of embodiments 52-53,wherein said antibody comprises SEQ ID NO: 21 and SEQ ID NO: 24.

Embodiment 55

The monoclonal antibody according to any one of the precedingembodiments, which is a humanized antibody.

Embodiment 56

The monoclonal antibody according to embodiment 55, in which frameworkregion 2 of the heavy chain comprises the amino acids:

-   -   T, in the position corresponding to position 40,    -   E, in the position corresponding to position 42,    -   R, in the position corresponding to position 44 and    -   A, in the position corresponding to position 49

of SEQ ID NO: 18. Embodiment 57

The monoclonal antibody according to embodiment 55, in which frameworkregion 2 of the heavy chain comprises the amino acids corresponding topositions 36 to 49 (WVRQTPEKRLEWVA) of SEQ ID NO: 18.

Embodiment 58

The monoclonal antibody according to any one of embodiments 1-54, whichis a human antibody.

Embodiment 59

The monoclonal antibody according to any one of embodiments 1-54, whichis a chimeric antibody

Embodiment 60

The monoclonal antibody according to any one of the preceedingembodiments, wherein the isotype of said antibody is IgG.

Embodiment 61

The monoclonal antibody according to embodiment 60, wherein said isotypeis IgG1, IgG2 or IgG4.

Embodiment 62

The monoclonal antibody according to embodiment 61, wherein the isotypeof said antibody is IgG4.

Embodiment 63

The monoclonal antibody according to any one of embodiments 60-62,wherein at least one amino acid of the Fc region of said antibody hasbeen substituted with another amino acid.

Embodiment 64

The monoclonal antibody according to any one of the precedingembodiments wherein the Fc region of said antibody is at least 80%, suchas at least 85%, such as at least 90%, such as at least 95%, such as95-100% identical amino acids 122-448 of SEQ ID NO: 24.

Embodiment 65

A monoclonal antibody, that is capable of binding the K2 domain of TFPIwith a higher affinity than mAb0281.

Embodiment 66

The monoclonal antibody, according to any one of embodiments 1-64, thatis capable of binding the K2 domain of TFPI with a higher affinity thanmAb0281.

Embodiment 67

A monoclonal antibody, that is capable of binding the K2 domain of TFPIwith a higher affinity than mAb4904.

Embodiment 68

A monoclonal antibody, according to any one of embodiments 1-66, that iscapable of binding the K2 domain of TFPI with a higher affinity thanmAb4904.

Embodiment 69

A monoclonal antibody, that is capable of binding the K2 domain of TFPIwith a higher affinity than mAb2974.

Embodiment 70

A monoclonal antibody, according to according to any one of embodiments1-68, that is capable of binding the K2 domain of TFPI with a higheraffinity than mAb2974.

Embodiment 71

A monoclonal antibody, that is capable of binding the K2 domain of TFPIwith a higher affinity than mAb29741.

Embodiment 72

A monoclonal antibody, according to according to any one of embodiments1-70, that is capable of binding the K2 domain of TFPI with a higheraffinity than mAb29741.

Embodiment 73

A monoclonal antibody, that is capable of binding the K2 domain of TFPI,wherein the K_(D) of said antibody is less than 0.8 nM, such as lessthan 0.7 nM, such as less than 0.6 nM, such as less than 0.5 nM, such asless than 0.4 nM, such as less than 0.3 nM, such as less than 0.2 nM,such as less than 0.1 nM, such as less than 0.05 nM, such as less than0.025 nM.

Embodiment 74

The monoclonal antibody, according to any one of embodiments 1-73,wherein the K_(D) of said antibody is less than 0.8 nM, such as lessthan 0.7 nM, such as less than 0.6 nM, such as less than 0.5 nM, such asless than 0.4 nM, such as less than 0.3 nM, such as less than 0.2 nM,such as less than 0.1 nM, such as less than 0.05 nM, such as less than0.025 nM.

Embodiment 75

The monoclonal antibody, according to any one of the above embodimentsthat is capable of binding the K2 domain of platelet-associated TFPI.

Embodiment 76

The monoclonal antibody, according to any of the above embodiments thatis capable of inhibiting soluble TFPI.

Embodiment 77

The monoclonal antibody according to embodiment 76, wherein said solubleTFPI may be completely inhibited.

Embodiment 78

The monoclonal antibody, according to any of the above embodiments thatis capable of inhibiting lipoprotein-bound TFPI.

Embodiment 79

The monoclonal antibody, according to any of the above embodiments thatis capable of inhibiting endothelial cell-bound TFPI.

Embodiment 80

A monoclonal antibody, that is capable of binding the K2 domain of TFPIsuch that FXa retains its activity by at least 91%, such as at least92%, such as at least 93%, such as at least 94%, such as at least 95%,such as at least 96%, such as at least 97%, such as at least 98%, suchas at least 99%, such as 99-100%, as measured in a FXa inhibition assay.

Embodiment 81

The monoclonal antibody, according to any one of embodiments 1-79, thatis capable of binding TFPI such that FXa retains its activity by atleast 91%, such as at least 92%, such as at least 93%, such as at least94%, such as at least 95%, such as at least 96%, such as at least 97%,such as at least 98%, such as at least 99%, such as 99-100% as measuredin a FXa inhibition assay.

Embodiment 82

A monoclonal antibody, that is capable of binding the K2 domain of TFPIsuch that the percentage of free TFPI in a subject is reduced to lessthan 30%, such as less than 29%, such as less than 28%, such as lessthan 27%, such as less than 26%, such as less than 25%, such as lessthan 24%, such as less than 23%, such as less than 22%, such as lessthan 21%, such as less than 20%, such as less than 19%, such as lessthan 18%, such as less than 17%, such as less than 16%, such as lessthan 15%, such as less than 14%, such as less than 13%, such as lessthan 12%, such as less than 11%, such as less than 10%, such as lessthan 9%, such as less than 8%, such as less than 7%, such as less than6%, such as less than 5%, such as less than 4%, such as less than 3%,such as less than 2%, such as less than 1%, such as between 1% and 0%.

Embodiment 83

A monoclonal antibody according to any one of claims 1-81, that iscapable of binding the K2 domain of TFPI such that the percentage offree TFPI in a subject is reduced to less than 30%, such as less than29%, such as less than 28%, such as less than 27%, such as less than26%, such as less than 25%, such as less than 24%, such as less than23%, such as less than 22%, such as less than 21%, such as less than20%, such as less than 19%, such as less than 18%, such as less than17%, such as less than 16%, such as less than 15%, such as less than14%, such as less than 13%, such as less than 12%, such as less than11%, such as less than 10%, such as less than 9%, such as less than 8%,such as less than 7%, such as less than 6%, such as less than 5%, suchas less than 4%, such as less than 3%, such as less than 2%, such asless than 1%, such as between 1% and 0%.

Embodiment 84

The monoclonal antibody according to embodiment 83, wherein the amountof free TFPI in a subject is reduced to said percentage during the first28 days, such as during the first 27 days, such as during the first 26days, such as during the first 25 days, such as during the first 24days, such as during the first 23 days, such as during the first 22days, such as during the first 21 days, such as during the first 20days, such as during the first 19 days, such as during the first 18days, such as during the first 17 days, such as during the first 16days, such as during the first 15 days, such as during the first 14days, such as during the first 13 days, such as during the first 12days, such as during the first 11 days, such as during the first 10days, such as during the first 9 days, such as during the first 8 days,such as during the first 7 days, such as during the first 6 days, suchas during the first 5 days, such as during the first 4 days, such asduring the first 3 days, such as during the first 2 days, such as duringthe first day after administration of said monoclonal antibody to saidindividual.

Embodiment 85

A monoclonal antibody, that is capable of binding the K2 domain of TFPIand that is capable of neutralising the TFPI inhibition ofmembrane-bound FVIIa/TF/FXa by at least 55%, such as at least 60%, suchas at least 65%, such as at least 70%, such as at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as up to 100%, such as 100%, as measured in an FVIIa/TF/FXainhibitor assay, when TFPI is saturated with said antibody.

Embodiment 86

The monoclonal antibody, according to any of embodiments 1-84, whereinsaid antibody is capable of neutralising the TFPI inhibition ofmembrane-bound FVIIa/TF/FXa by at least 55%, such as at least 60%, suchas at least 65%, such as at least 70%, such as at least 75%, such as atleast 80%, such as at least 85%, such as at least 90%, such as at least95%, such as up to 100%, such as 100%, as measured in an FVIIa/TF/FXainhibitor assay, when TFPI is saturated with said antibody.

Embodiment 87

A monoclonal antibody that is capable of binding the K2 domain of TFPIand that reduces in vivo clotting time without significantly reducingthe platelet count.

Embodiment 88

The monoclonal antibody, according to any one of embodiments 1-86,wherein said antibody reduces in vivo clotting time withoutsignificantly reducing the platelet count.

Embodiment 89

The monoclonal antibody, according to embodiments 88, wherein saidplatelet count does not fall to approximately 80%, such as approximately75%, such as approximately 70%, such as approximately 65%, such asapproximately 60%, such as approximately 55%, such as approximately 50%,such as approximately 45%, such as approximately 40%, such asapproximately 35%, such as approximately 30%, such as approximately 25%of the original platelet count.

Embodiment 90

A monoclonal antibody that is capable of binding the K2 domain of TFPIand that reduces in vivo clotting time without causing transientthrombocytopaenia.

Embodiment 91

The monoclonal antibody, according to any one of embodiments 1-89,wherein said antibody reduces in vivo clotting time without causingtransient thrombocytopaenia.

Embodiment 92

A fragment of the monoclonal antibody according to any one of thepreceding embodiments.

Embodiment 93

The fragment according to embodiment 92, which is a Fab fragment, aF(ab′)₂ fragment, a Fab′ fragment, a Fd fragment, a Fv fragment or a dAbfragment.

Embodiment 94

A variant of the monoclonal antibody according to any one ofembodiments, which is a deletion variant or an insertion variant.

Embodiment 95

A pharmaceutical formulation comprising the monoclonal antibodyaccording to any one of embodiments 1-94.

Embodiment 96

A pharmaceutical formulation comprising the monoclonal antibodyaccording to any one of embodiments 1-94, wherein said formulation issuitable for parenteral use.

Embodiment 97

A pharmaceutical formulation comprising the monoclonal antibodyaccording to any one of embodiments 1-94, wherein said antibody issuitable for intravenous use.

Embodiment 98

A pharmaceutical formulation comprising the monoclonal antibodyaccording to any one of embodiments 1-94, wherein said antibody issuitable for intramuscular use.

Embodiment 99

A pharmaceutical formulation comprising the monoclonal antibodyaccording to any one of embodiments 1-94, wherein said antibody issuitable for subcutaneous use.

Embodiment 100

Use of the monoclonal antibody according to any one of embodiments 1-94for the manufacture of a medicament suitable for parenteraladministration.

Embodiment 101

Use of the monoclonal antibody according to any one of embodiments 1-94for the manufacture of a medicament suitable for intravenousadministration.

Embodiment 102

Use of the monoclonal antibody according to any one of embodiments 1-94for the manufacture of a medicament suitable for intramuscularadministration.

Embodiment 103

Use of the monoclonal antibody according to any one of embodiments 1-94for the manufacture of a medicament suitable for subcutaneousadministration.

Embodiment 104

Use of a monoclonal antibody according to any one of embodiments 1-94,for the treatment of a subject with a coagulopathy.

Embodiment 105

Use according to embodiment 104, wherein said subject has anycongenital, acquired and/or iatrogenic coagulopathy, such as may beselected from the group consisting of haemophilia A, with or withoutinhibitors, and haemophilia B, with or without inhibitors.

Embodiment 106

Use according to any one of embodiments 95-105, wherein said monoclonalantibody significantly reduces blood loss.

Embodiment 107

Use according to any one of embodiments 95-106, wherein said monoclonalantibody significantly reduces bleeding time.

Embodiment 108

Use according to any one of embodiments 95-107, wherein the amount ofmonoclonal antibody administered results in a plasma concentration ofabout 10 μg/ml to about 40 μg/ml, such as about 15-35 μg/ml, such asabout 10-15 μg/ml, such as about 15-20 μg/ml, such as about 20-25 μg/ml,such as about 25-30 μg/ml, such as about 30-35 μg/ml, such as about35-40 μg/ml, of said monoclonal antibody.

Embodiment 109

A method of treating a subject with a coagulopathy, comprisingadministering to said subject the monoclonal antibody according to anyone of embodiments 1-94.

Embodiment 110

The method according to embodiment 109, wherein said coagulopathy is anycongenital, acquired and/or iatrogenic coagulopathy, such as may beselected from the group consisting of haemophilia A, with or withoutinhibitors, and haemophilia B, with or without inhibitors.

Embodiment 111

The method according to any one of embodiments 109-110, wherein saidmonoclonal antibody is capable of significantly reducing blood loss.

Embodiment 112

The method according to any one of embodiments 109-111, wherein saidmonoclonal antibody is capable of significantly reducing bleeding time.

Embodiment 113

The method according to any one of embodiments 109-112, wherein theamount of monoclonal antibody administered is such as to saturate itstarget.

Embodiment 114

The method according to any one of embodiments 109-113, wherein theamount of monoclonal antibody administered is such as to saturatesoluble TFPI.

Embodiment 115

The method according to any one of embodiments 109-114, wherein saidadministered antibody is capable of completely inhibiting soluble TFPI.

Embodiment 116

The method according to any one of embodiments 109-115, wherein saidmonoclonal antibody is administered in an amount sufficient to saturateendothelium-bound TFPI.

Embodiment 117

The method according to any one of embodiments 109-116, wherein theamount of monoclonal antibody administered results in a plasmaconcentration of about 10 μg/ml to about 40 μg/ml, such as about 15-35μg/ml, such as about 10-15 μg/ml, such as about 15-20 μg/ml, such asabout 20-25 μg/ml, such as about 25-30 μg/ml, such as about 30-35 μg/ml,such as about 35-40 μg/ml, of said monoclonal antibody.

Embodiment 118

The method according to any one of embodiments 109-117, wherein a singledose may be administered.

Embodiment 119

The method according to any one of embodiments 109-118, wherein multipledoses may be administered.

Embodiment 120

The method according to any one of embodiments 109-119, wherein saidantibody may be administered daily.

Embodiment 121

The method according to any one of embodiments 109-120, wherein saidantibody may be administered every other day.

Embodiment 122

The method according to any one of embodiments 109-121, wherein saidantibody may be administered every third day.

Embodiment 123

The method according to any one of embodiments 109-122, wherein saidantibody may be administered every fourth day.

Embodiment 124

The method according to any one of embodiments 109-123, wherein saidantibody may be administered every fifth day.

Embodiment 125

The method according to any one of embodiments 109-124, wherein saidantibody may be administered every sixth day.

Embodiment 126

The method according to any one of embodiments 109-125, wherein saidmonoclonal antibody may be administered approximately every week, suchas every 5, 6, 7, 8, 9 or 10 days.

Embodiment 127

The method according to any one of embodiments 109-126, wherein saidmonoclonal antibody may be administered approximately every other week,such as every 11, 12, 13, 14, 15, 16 or 17 days.

Embodiment 128

The method according to any one of embodiments 109-127, wherein saidmonoclonal antibody may be administered approximately every third week,such as every 18, 19, 20, 21, 22, 23 or 24 days.

Embodiment 129

The method according to any one of embodiments 109-128, wherein saidmonoclonal antibody may be administered approximately every fourth week,such as every 25, 26, 27, 28, 29, 30 or 31 days.

Embodiment 130

The method according to any one of embodiments 109-129, wherein thedosage may be approximately 0.1-10 mg/kg, such as approximately 0.1-1mg/kg, such as approximately 1-2 mg/kg or approximately 2-3 mg/kg orapproximately 4-5 mg/kg or approximately 5-6 mg/kg or approximately 6-7mg/kg or approximately 7-8 mg/kg or approximately 8-9 mg/kg orapproximately 9-10 mg/kg; or approximately 10-21 mg/kg, such asapproximately 10-11 mg/kg, or approximately 11-12 mg/kg, orapproximately 12-13 mg/kg, or approximately 13-14 mg/kg, orapproximately 14-15 mg/kg, or approximately 15-16 mg/kg, orapproximately 16-17 mg/kg, or approximately 17-18 mg/kg, orapproximately 18-19 mg/kg, or approximately 19-20 mg/kg or approximately20-21 mg/kg.

Embodiment 131

The method according to any one of embodiments 109-130, wherein thedosage may be approximately 2 to 200 mg/kg, such as about 150-200 mg/kg,such as about 150-170 mg/kg, such as about 100-150 mg/kg, such as about50-100 mg/kg, such as about 70-90 mg/kg, such as about 10-50 mg/kg, suchas about 10-30 mg/kg.

Embodiment 132

The method according to any one of embodiments 109-131, wherein saidmonoclonal antibody may be administered parenterally.

Embodiment 133

The method according to embodiment 132, wherein said monoclonal antibodymay be administered intravenously.

Embodiment 134

The method according to embodiment 133, wherein the dosage of saidmonoclonal antibody may be approximately 10-20 mg/kg.

Embodiment 135

The method according to any one of embodiments 133-134, wherein themonoclonal antibody may be administered every other week.

Embodiment 136

The method according to any one of embodiments 133-135, wherein themonoclonal antibody may be administered every third week.

Embodiment 137

The method according to any one of embodiments 133-136, wherein themonoclonal antibody may be administered every fourth week.

Embodiment 138

The method according to embodiment 133, wherein the dosage of saidmonoclonal antibody may be approximately 10-20 mg/kg and said monoclonalantibody may be administered every other week.

Embodiment 139

The method according to embodiment 133, wherein the dosage of saidmonoclonal antibody may be approximately 10-20 mg/kg and said monoclonalantibody may be administered every third week.

Embodiment 140

The method according to embodiment 133, wherein the dosage of saidmonoclonal antibody may be approximately 10-20 mg/kg and said monoclonalantibody may be administered every fourth week.

Embodiment 141

The method according to embodiment 132, wherein said monoclonal antibodymay be administered intramuscularly.

Embodiment 142

The method according to embodiment 132, wherein said monoclonal antibodymay be administered subcutaneously.

Embodiment 143

The method according to embodiment 132, wherein the dosage of saidmonoclonal antibody may be approximately 1 mg/kg

Embodiment 144

The method according to any one of embodiments 141-143, wherein themonoclonal antibody may be administered daily.

Embodiment 145

The method according to any one of embodiments 141-144, wherein themonoclonal antibody may be administered every other day.

Embodiment 146

The method according to any one of embodiments 141-145, wherein thedosage of said monoclonal antibody may be approximately 1 mg/kg andwherein said monoclonal antibody may be administered daily.

Embodiment 147

The method according to embodiment any one of embodiments 141-146,wherein the dosage of said monoclonal antibody may be approximately 1mg/kg and wherein said monoclonal antibody may be administered everyother day.

Embodiment 148

The method according to any one of embodiments 109-147, wherein saidantibody may be administered prophylactically.

Embodiment 149

The method according to any one of embodiments 109-148, wherein saidantibody may be administered therapeutically (on demand).

Embodiment 150

The method according to any one of embodiments 109-149, wherein saidadministered antibody is capable of completely (100%) inhibiting solubleTFPI.

Embodiment 151

A polynucleotide encoding the monoclonal antibody according to any oneof embodiments 1-94.

Embodiment 152

A polynucleotide according to embodiment 151, which comprises at leastone sequence selected from the group consisting of SEQ ID NOs: 13, 16,19 and 22.

Embodiment 153

A polynucleotide according to embodiment 152, which comprises SEQ ID NO:19.

Embodiment 154

A polynucleotide according to embodiment 152, which comprises SEQ ID NO.22.

Embodiment 155

A polynucleotide according to embodiment 152, which comprises SEQ IDNOs: 19 and 22.

Embodiment 156

A eukaryotic cell which comprises the polynucleotide according to anyone of embodiments 151-155.

Embodiment 157

A eukaryotic cell which expresses the monoclonal antibody, or fragmentthereof, according to any one of embodiments 1-94.

Embodiment 158

The eukaryotic cell according to embodiment 157, which is a mammaliancell.

Embodiment 159

The eukaryotic cell according to embodiment 157, which is a yeast cell.

Embodiment 160

The mammalian cell according to embodiment 158, which is selected fromthe group consisting of HEK293, CHO, BHK, NSO and human retina cells.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

Example 1 Production and Characterisation of Monoclonal AntibodiesDirected Against TFPI

Monoclonal antibodies were generated against tissue factor pathwayinhibitor (TFPI). A monoclonal antibody having the desired bindingspecificity was identified, cloned and sequenced. This antibody wasfound to significantly reduce cuticle bleeding time in vivo and to leadto no significant drop in platelet number.

Methods and Results

All kits were used according to the manufacturers' instructions.Abbreviations: HC: heavy chain; LC: light chain; VH: variabledomain—heavy chain; VL: variable domain-light chain; PCR: polymerasechain reaction.

Immunisation and Fusion

Mice were immunized with both full length TFPI and the short versionTFPIB161B which contains only the first two Kunitz domains. RBF micewere used for immunizations and production of mouse monoclonalantibodies. Injections were made subcutaneously in the back of the mice.20 μg protein was mixed with complete Freund's adjuvant for the firstinjection. In the subsequent immunizations, incomplete Freund's adjuvantwas used with same concentration of the antigen. Ten days after the lastimmunization, eye-blood from mice was screened by ELISA for TFPIspecific antibodies. Mice with positive serum titres were boosted with10 μg of TFPI by intravenous injection, and sacrificed after three days.The spleens were removed aseptically and dispersed to a single cellsuspension. Fusion of spleen cells and myeloma cells was done by thePEG-method or by electrofusion.

Binding Assay: ELISA

Immunoplates were coated with anti-mouse IgG. Culture supernatants fromthe hybridoma cells were added to the plates and, after washing, solublebiotinylated human TFPI or TFPIB161B was added to test for specificbinding.

Neutralizing Assays: FXa Assay and TF/FVIIa/FXa Assay

FXa inhibition assay: a fixed concentration of TFPI giving rise to 90%inhibition of FXa was pre-incubated with culture supernatants fromhybridoma cells containing anti TFPI monoclonal antibodies and added toFXa plus FXa-specific chromogenic substrate. This assay addresses TFPIbinding to FXa (described in greater detail in example 6).

FVIIa/TF/FXa inhibition assay: 1) Incubation of culture supernatantsfrom hybridoma cells containing anti TFPI monoclonal antibodies anti andfixed TFPI (90% inhibition of FVIIa/TF); 2) Incubation ofTFPI+FVIIa+TF+FXa; 3) Addition of FX (FX>>FXa) followed by incubationwith FXa chromogenic substrate (described in greater detail in example7).

Dilute Prothrombin Time (dPT)

A dilute Prothrombin (PT) analysis: human plasma in combination withdiluted human thromboplastin (TF source). Clot time in the plasma wasmeasured upon addition of increasing protein A purified TFPI monoclonalantibody concentrations to look for dose dependent reduction of clottingtime. FVIIa (25 nM) was the positive control and must shorten this clottime.

Binding Interaction Analysis

Binding interaction analysis was obtained by Surface Plasmon Resonancein a Biacore 3000. Capture of the relevant monoclonal antibody at afixed concentration was obtained with immobilised mouse anti-IgG.Different concentrations of TFPI were tested. Determination of bindingconstants (k_(on), k_(ap) K_(D)) was obtained assuming a 1:1 interactionof TFPI and the antibody of interest (described in greater detail inexample 8).

Thrombelastography

This records the kinetic of clot formation and fibrinolysis in wholeblood. Haemophilia A-like condition is induced by pre-incubating theblood with neutralizing anti-FVIII IgG.

Antibody Cloning and Sequencing

Murine heavy chain and light chain sequences for an anti-TFPI antibodywere cloned from a hybridoma: TFPI-4F36A1B2 (abbreviated herein to4F36). Total RNA, extracted from hybridoma cells using the RNeasy-MiniKit from Qiagen, was used as templates for cDNA synthesis. cDNA wassynthesized in a 5′-RACE reaction using the SMART™ RACE cDNAamplification kit from Clontech. Subsequent target amplification of HCand LC sequences was performed by PCR using Phusion Hot Star polymerase(Finnzymes) and the universal primer mix (UPM) included in the SMART™RACE kit as a forward primer. A reverse primer with the followingsequence was used for HC (VH domain) amplification:5′-CCCTTGACCAGGCATCCCAG-3′ (primer #129). A reverse primer with thefollowing sequence was used for LC amplification:5′-GCTCTAGACTAACACTCATTCCTGTTGAAGCTCTTG-3′ (primer #69).

PCR products were separated by gel electrophoresis, extracted using theGFX PCR DNA and Gel Band Purification Kit from GE HealthcareBio-Sciences and cloned for sequencing using a Zero Blunt TOPO PCRCloning Kit and chemically competent TOP10 E. coli from Invitrogen.Colony PCR was performed on selected colonies using an AmpliTaq GoldMas-ter Mix from Applied Biosystems and M13uni/M13rev primers. ColonyPCR clean-up was performed using the ExoSAP-IT enzyme mix (usb).Sequencing was performed at MWG Biotech, Martinsried Germany usingeither M13uni(−21)/M13rev(−29) or T3/T7 sequenc-ing primers. Sequenceswere analyzed and annotated using the Vector NTI program.

From hybridoma TFPI-4F36A1B2 a single unique murine kappa type LC wasidentified and a single unique murine HC, subclass IgG1. LC sequence isgiven in SEQ ID NO: 6 and HC sequence is given in SEQ ID NO: 10. VH & VLSequences are shown in FIG. 2, leader peptide sequences are notincluded.

Epitopes

TFPI1 includes three Kunitz domains (see FIG. 4). Surface accessibleresidues of the Kunitz domains of TFPI1 were identified from existingstructures of TFPI1-2. In particular, residues with a relativeaccessibility larger than 40% are considered to be surface accessible.For TFPI1-2 this comprises (see FIG. 5): amino acids 94-95, 98, 100-110,118-121, 123-124, 131, 134, 138-142 and 144-145.

Example 2 Cloning and Sequencing of Mouse TFPI4F36A1B2 mAb

This example describes cloning and sequencing of the murine heavy chainand light chain sequences of anti-TFPI antibody: TFPI4F36A1B2. Total RNAwas extracted from hybridoma cells using the RNeasy-Mini Kit from Qiagenand used as template for cDNA synthesis. cDNA was synthesized in a5′-RACE reaction using the SMART™ RACE cDNA amplification kit fromClontech. Subsequent target amplification of HC and LC sequences wasperformed by PCR using Phusion Hot Start polymerase (Finnzymes) and theuniversal primer mix (UPM) included in the SMART™ RACE kit as forwardprimer. The reverse primer identified as SEQ ID NO: 11 was used for HC(VH domain) amplification and the reverse primer identified as SEQ IDNO: 12 was used for LC amplification. PCR products were separated by gelelectrophoresis, extracted using the GFX PCR DNA & Gel Band PurificationKit from GE Healthcare Bio-Sciences and cloned for sequencing using aZero Blunt TOPO PCR Cloning Kit and chemically competent TOP10 E. coli(Invitrogen). Colony PCR was performed on selected colonies using anAmpliTaq Gold Master Mix from Applied Biosystems and M13uni/M13revprimers. Colony PCR clean-up was performed using the ExoSAP-IT enzymemix (USB). Sequencing was performed at MWG Biotech, Martinsried Germanyusing either M13uni(−21)/M13rev(−29) or T3/T7 sequencing primers.Sequences were analyzed and annotated using the Vector NTI program. Allkits and reagents were used according to the manufacturer'sinstructions.

A single unique murine kappa type LC and a single unique murine HC,subclass IgG1 was identified. The nucleic acid and amino acid sequencesfor the variable light chain are shown in SEQ ID NOs: 3 and 5,respectively. The nucleic acid and amino acid sequences for the variableheavy chain are shown in SEQ ID NOs: 7 and 9, respectively. Leaderpeptide sequences are not included in these sequences.

BLAST Searches

The translated anti-TFPI4F36A1B2 VL and VH amino acid sequences wereused as query sequences. BLAST searches were performed against sequencesin the Uniprot database using the BLASTp translations program. Theoutput for the anti-TFPI4F36A1B2 VH produces alignments of which >20 ofthe 50 highest identity scores were murine Ig heavy chain sequences. Thehighest identity scores were 81% (99/121) against a mouse Ig heavychain. The output for the anti-TFPI4F36A1B2 VL produces alignments ofwhich >30 of the 50 highest identity scores were murine Ig kappa lightchain sequences. The highest identity score was 92% (105/113) against amouse Ig kappa light chain. In conclusion, the VH and VL sequences foranti-TFPI4F36A1B2 represent new unique sequences.

Generation of Mouse Anti-TFPI4F36A1B2 Expression Vectors

A series of CMV promotor-based based expression vectors (pTT vectors)were generated for transient expression of the mouse TFPI4F36 antibodyin the HEK293-6E EBNA-based expression system developed by Yves Durocher(Durocher et al. Nucleic Acid Research, 2002). In addition to the CMVpromotor, the vectors contain a pMB 1 origin, an EBV origin and the Ampresistance gene.

The region corresponding to the full length anti-TFPI4F36A1B2 LC(including the original signal peptide sequence) was PCR amplified fromthe original TOPO sequencing clones using primers specific for the N andC-terminal sequences. The sense primer contained a terminal HindIIIrestriction site sequences for cloning purposes and a Kozak sequence(5′-GCCGCCACC-3′) immediately upstream of the ATG start codon. Theanti-sense primer contained a stop codon followed by an XbaI restrictionsite sequence, immediately downstream of the coding sequence. Thegenerated PCR fragment was restriction digested, cloned into themultiple cloning site (MCS) of a linearized pTT-based vector andtransformed into E. coli for selection. The sequence of the finalconstruct was verified by DNA sequencing.

The region corresponding to the VH domain (including the original signalpeptide sequence) was PCR amplified from the original TOPO sequencingclones using primers specific for the N-terminal sequence and VH/CHtransition sequence. The sense primer contained a terminal NotIrestriction site sequences for cloning purposes and a Kozak sequence(5′-GCCGCCACC-3′) immediately upstream of the ATG start codon. Theanti-sense primer contained an in-frame NheI restriction site downstreamof the VH/CH transition. The generated VH domain PCR fragment wasrestriction digested, cloned into a linearized vector containing the CHdomain sequence for a murine IgG1 and transformed into E. coli forselection. The sequence of the final construct was verified by DNAsequencing.

The cloned and recombinantly expressed anti-TFPI4F36A1B2 antibody hadthe same profile and affinity in all assay used, as the originalhybridoma derived antibody. Procedures used for transient expression inHEK293-6E cells are described in example 3.

Example 3 Design and Construction of a Humanized TFPI4F36 mAb

The mouse anti-TFPI4F36A1B2 CDR sequences were annotated according tothe Kabat definition and found to be as follows:

CDR-H1: (amino acids 31-35 of SEQ ID NO: 8) NYAMS. CDR-H2:(amino acids 50-66 of SEQ ID NO: 8) TISRSGSYSYFPDSVQG. CDR-H3:(amino acids 99-110 of SEQ ID NO: 8) LGGYDEGDAMDS. CDR-L1:(amino acids 24-39 of SEQ ID NO: 4) KSSQSLLESDGKTYLN. CDR-L2:(amino acids 55-61 of SEQ ID NO: 4) LVSILDS. CDR-L3:(amino acids 94-102 of SEQ ID NO: 4) LQATHFPQT.

A 3D model of anti-TFPI4F36A1B2 was built in Modeller(wwww.salilab.org/modeller/) based on the structural templates 2GJJ (mABagainst Her2erbb2) and 1X9Q (hAB against flourescein).

A BLASTp search in a human germline V database with theanti-TFPI4F36A1B2 VL and VH returned the following four potentialgermline sequences:

Heavy chain: VH3_(—)21 or VH7183.9 (E-values <1e-45)Light chain: VKII_A18 or VKII_A1 (E-values <3e-45)

After manual inspection of hits and alignments, the VH3_(—)21 andVKII_A18 germline sequences were selected as HC and LC humanizationframeworks, respectively. The corresponding germline J-segments wereselected based on sequence alignment as JH6 and JK4. The alignmentbetween anti-TFPI4F36A1B2 and the selected germline sequences are shownin combination with the first CDR grafted version of the humanizedTFPI4F36. The sequence identity between anti-TFPI4F36A1B2 and the humanscaffolds (HC: VH3_(—)21/JH6 and LC: VKII_A18/JK4) is very high asillustrated by asterisks below the sequence. Each asterisk marks aposition of sequence identity. The initial humanized VH construct wasdesigned according to a minimal CDR grafting strategy, in which CDR-H2is grafted in a shorter version (residue 50-58) than the Kabatdefinition (residue 50-66). The remaining 5 CDRs were grafted accordingto the Kabat definition. The CDRs (Kabat definition) are listed asgrafted below; the residues shown in bold for CDR-H2 are human germlineresidues.

CDR-H1: (amino acids 31-35 of SEQ ID NO: 18) NYAMS. CDR-H2:(amino acids 50-66 of SEQ ID NO: 28) TISRSGSYSYYADSVKG. CDR-H3:(amino acids 99-110 of SEQ ID NO: 18) LGGYDEGDAMDS. CDR-L1:(amino acids 24-39 of SEQ ID NO: 15) KSSQSLLESDGKTYLN. CDR-L2:(amino acids 55-61 of SEQ ID NO: 15) LVSILDS. CDR-L3:(amino acids 94-102 of SEQ ID NO: 15) LQATHFPQT.

The composition of CDR-H2 in the final humanized variant HzTFPI4F36 islisted below and matched the CDR-H2 listed for the mouse antibodyanti-TFPI4F36A1B2.

CDR-H2: (amino acids 50-66 of SEQ ID NO: 18) TISRSGSYSYFPDSVQG.

FIG. 1 shows the sequences of VH (A) and VL (B) domains of mouseanti-TFPI4F36A1B2 (SEQ ID NOs: 8 and 4, respectively) aligned with humangermline sequences (SEQ ID NOs: 32 and 31, respectively) and the CDRgrafted humanized TFPI4F36 sequences (SEQ ID NOs: 28 and 26,respectively). The Kabat numbering scheme is used, as shown above thesequences in the figure, and CDRs according to the Kabat definition areshown in bold. Differences in the framework regions between the mouseanti-TFPI4F36A1B2 and the germline sequences are highlighted in grey inthe anti-TFPI4F36A1B2 sequence. Asterisks indicate positions of sequenceidentity between the mouse TFPI4F36 and human germline sequences.Potential back mutations are highlighted in gray in theHzTFPI4F36-CDRgrafted sequence (listed as hz4F36CDRgraft).

Potential back mutations for the HzTFPI4F36-CDRgrafted constructs wereidentified based on the positional differences found in the frameworksregions of mouse TFPI4F36 and the germline sequence. A 3D FIG. 1 showsthe sequences model of the TFPI4F36 Fab fragment was also used toidentify and prioritize potential back mutations. The lists of generatedback mutations in the humanized TFPI4F36 LC and HC are shown in tables 2and 3, respectively.

Generation of Expression Vectors for Humanized TFPI4F36

DNA sequences for humanized TFPI4F36 VH and VL regions were synthesized(GENEART AG) according to the humanization design of the antibodydescribed above. The sequences were obtained with the basic minimal CDRgrafting and no additional back mutations. The respective LC and HCgermline leader peptide sequences were include in the constructs as wellas a Kozak sequence (5′-GCCGCCACC-3′) immediately upstream of the ATGstart codon.

pTT-based expression vectors were generated for transient expression ofthe humanized TFPI4F36 antibody as a human kappa/IgG4(S241P) isotype.The proline mutation at position 241 (numbering according to Kabat,corresponding to residue 228 per the EU numbering system (Edelman G.M.et A L., Proc. Natl. Acad. USA 63, 78-85 (1969)) was introduced in theIgG4 hinge region to eliminated formation of monomeric antibodyfragments, i.e. “half-antibodies” comprising of one LC and one HC.

The VH fragment was excised from the GENEART cloning vector and clonedinto a linearized pTT-based vector containing the sequence for a humanIgG4(S241P) CH domain subsequently transformed into E. coli forselection. The sequence of the final construct was verified by DNAsequencing. The VL fragment was excised from the GENEART cloning vectorand cloned into a linearized pTT-based vector containing the sequencefor a human kappa CL domain and subsequently transformed into E. colifor selection. The sequence of the final construct was verified by DNAsequencing.

Nucleic acid and amino acid sequences for the VL, VH, LC and HC of theCDR-grafted HzTFPI4F36 monoclonal antibody (signal peptide sequenceomitted) are provided in the sequence listing (SEQ ID NOs: 26-30).

Generation of Expression Vectors for Mouse/Human Chimeric TFPI4F36

To enable the best possible evaluation of the humanized TFPI4F36variants, a mouse/human chimera version of the anti-TFPI4F36 antibody(ChimTFPI4F36) was constructed in order to eliminate any differencesrelated to constant region origin and isotype. pTT-based expressionvectors were generated for transient expression of chimericanti-TFPI4F36 antibody with murine variable domains on the humankappa/IgG4(S241P) isotype scaffolds.

The region corresponding to the VH domain was PCR amplified from aanti-TFPI4F36A1B2 HC expression plasmid using a generic pTT specificprimer and a primer specific for the VH domain C-terminus The senseprimer is specific for at sequence stretch upstream of the HindIIIrestriction site and the ATG start codon. The anti-sense primercontained an in-frame NheI restriction site in the VH/CH transitionsequence. The generated PCR fragment was restriction digested, clonedinto a linearized pTT-based vector containing the sequence for a humanIgG4(S241P) CH domain and subsequently transformed into E. coli forselection. The sequence of the final construct was verified by DNAsequencing.

The region corresponding to the VL domain was PCR amplified from aTFPI4F36A1B2 LC expression plasmid using a generic pTT specific primerand a primer specific for the VL domain C-terminus. The sense primer isspecific for at sequence stretch upstream of the HindIII restrictionsite and the ATG start codon. The anti-sense primer contained anin-frame BsiWI restriction site in the VL/CL transition sequence. Thegenerated PCR fragment was restriction digested, cloned into alinearized pTT-based vector containing the sequence for a human kappa CLdomain and subsequently transformed into E. coli for selection. Thesequence of the final construct was verified by DNA sequencing.

Recombinant Expression of mAb Variants

The murine anti-TFPI4F36A1B2, chimeric anti-TFPI4F36 and humanizedTFPI4F36 antibody variants were expressed transiently in HEK293-6E cellsfollowing a generic antibody expression protocol. The followingprocedure describes the generic transfection protocol used forsuspension adapted HEK293-6E cells.

Cell Maintenance

HEK293-6E cells were grown in suspension in FreeStyle™ 293 expressionmedium (Gibco) supplemented with 25 mg/ml Geneticin (Gibco), 0.1% v/v ofthe surfactant Pluronic F-68 (Gibco) & 1% v/v Penicillin-Streptomycin(Gibco). Cells were cultured in Erlenmeyer shaker flasks in shakerincubators at 37° C., 8% CO₂ and 125 rpm and maintained at celldensities between 0.1−1.5×10⁶ cells/ml.

DNA Transfection

-   -   The cell density of cultures used for transfection was        0.9−2.0×10⁶ cells/ml.    -   A mix of 0.5 μg LC vector DNA+0.5 μg HC vector DNA was used per        ml cell culture.    -   The DNA was diluted in Opti-MEM media (Gibco) 30u1 media/μg DNA,        mixed and incubated at room temperature (23-25° C.) for 5 min.    -   293Fectin™ (Invitrogen) was used as transfection reagent at a        concentration of 1 μl per μg DNA.    -   The 293Fectin™ was diluted 30× in Opti-MEM media (Gibco), mixed        and incubated at room temperature (23-25° C.) for 5 min.    -   The DNA and 293Fectin solutions were mixed and left to incubate        at room temperature (23-25° C.) for 25 min.    -   The DNA-293Fectin mix was then added directly to the cell        culture.    -   The transfected cell culture was transferred to a shaker        incubator at 37° C., 8% CO₂ and 125 rpm.    -   3-6 days post transfection, cell culture supernatants were        harvested by centrifugation, followed by filtration through a        0.22 μm PES filter (Corning).    -   Quantitative analysis of antibody production was performed by        Biolayer Interferometry directly on clarified cell culture        supernatants using the ForteBio Octet system and protein A        biosensors or quantitative protein A HPLC.

Activity Analyses of the CDR Grafted Variant of Humanized Anti-TFPI4F36

Humanization by minimal CDR grafting resulted in a dramatic loss ofaffinity caused by effect on both on- and off-rate. The TFPI bindingaffinity of the initially grafted version of the humanized TFPI4F36antibody (HzTFPI4F36-CDRgrafted, in table 1 listed as HumanizedTFPI4F36) was at least 100-fold lower than the ˜30 pM affinity of theoriginal mouse TFPI4F36 antibody (see table 1). Retention of affinity inthe chimeric antibody confirmed that the human kappa/IgG4(S241P) FC hadno effect on antibody affinity. The affinity analyses were done usingSRP as described below.

TABLE 1 mAb ka (1/Ms) kd (1/M) KD (M) Murine TFPI4F36 4.70E+06 1.33E−042.82E−11 Chimeric TFPI4F36 8.88E+06 1.44E−04 1.62E−11 Humanized TFPI4F361.07E+06 2.21E−03 2.06E−09Surface Plasmon Resonance (Biacore) Analysis of hzTFPI4F36-TFPIInteraction

The kinetic parameters for the interaction of recombinant human TFPI tothe original murine anti-TFPI4F36A1B2, chimeric anti-TFPI4F36, andvarious variants of the humanized TFPI4F36 antibody were determined bySPR analysis in Biacore, using two different approaches. Initialkinetics ranking studies were based on a capture procedure of purifiedmAbs as described in example 1. These were followed by a direct bindingkinetic procedure on selected mAb constructs, with the monoclonalantibody covalently coupled via free amine groups to thecarboxymethylated dextrane membrane (CM5) on the sensor chip surface.Recombinant human TFPI was injected in various concentrations, followedby a dissociation period with constant buffer flow over the sensor chipsurface as described in example 8.

Site-Directed Mutagenesis to Introduce Back Mutations in Humanized mAb

Based on the low affinity of the CDR grafted version of humanizedanti-TFPI4F36, a series of 27 human-to-mouse reverse mutations (referredto as back mutations) was generated in the light chain (LC) and heavychain (HC) of HzTFPI4F36-CDRgrafted. These mutants were expressed,purified and analyzed by Biacore, either as separate mutants or as LC/HCcombination mutants. The lists of generated mutations are shown intables 2 and 3, respectively.

Site-directed mutagenesis was performed to introduce human-to-mousereverse mutations (henceforth referred to as back mutations) at thespecific residues in the HzTFPI4F36-CDRgrafted LC/HC constructs ashighlighted in the humanization design. Mutations were introduced by twodifferent methods:

-   -   1) QuickChange® Site-Directed or Multi Site-Directed Mutagenesis        kits from Stratagene were used to introduce point mutations and        combination mutations. The kits were used according to the        manufacturer's protocol.    -   2) Standard 2-step overlapping PCR methods were also used to        introduce point mutations and to generate combination mutations.

The LC and HC expression plasmids for HzTFPI4F36-CDRgrafted were used astemplates for the first rounds of mutagenesis. In subsequent rounds,mutations were also introduces using previously mutated plasmids astemplate. The sequences of all final constructs were verified by DNAsequencing.

TABLE 2 Mutated variants of the HzTFPI4F36-CDRgrafted light chain LCmutants Mutations K_(D) (M) HzTFPI4F36 LC-S63T S63T 7.8E−9  HzTFPI4F36LC-P15I P15I 17.0E−9  HzTFPI4F36 LC-FR2 Y36L, K39R, Q42E, Q45K 6.3E−10HzTFPI4F36 LC-P15I, FR2 P15I, Y36L, K39R, Q42E, Q45K 6.4E−10 HzTFPI4F36LC-Y36L Y36L >3E−11 HzTFPI4F36 LC-K39R, K39R, Q42E, Q45K >3E−11 Q42E,Q45K

TABLE 3 Mutated variants of the HzTFPI4F36-CDRgrafted heavy chain HCmutants Mutations K_(D) (M) HzTFPI4F36 HC-Q3E Q3E 5.8E−9  HzTFPI4F36HC-G44R G44R 2.3E−9  HzTFPI4F36 HC-S49A S49A 3.0E−9  HzTFPI4F36 HC-Y59FY59F 5.5E−9  HzTFPI4F36 HC-A60P A60P 2.2E−9  HzTFPI4F36 HC-K64Q K64Q2.5E−9  HzTFPI4F36 HC-S77T S77T 1.5E−9  HzTFPI4F36 HC-A93T A93T 2.7E−9 HzTFPI4F36 HC-Y59F, Y59F, A60P 2.3E−9  A60P HzTFPI4F36 HC- Y59F, A60P,K64Q 9.0E−10 KABAT CDR2 HzTFPI4F36 HC-FR2, A40T, G42E, G44R, S49A1.3E−9  S49A HzTFPI4F36 HC-FR3 N82aS, A84S, V89M 5.3E−9  HzTFPI4F36HC-FR3, S77T, N82aS, A84S, V89M 7.7E−9  S77T HzTFPI4F36 HC-FR3, N82aS,A84S, V89M, A93T 4.1E−9  A93T HzTFPI4F36 HC-FR2 A40T, G42E, G44R 8.8E−10HzTFPI4F36 HC-FR2, A40T, G42E, G44R, S49A, Y59F, 2.6E−11 S49A, CDR2A60P, K64Q HzTFPI4F36 HC-G42E, G42E, G44R, Y59F, A60P, K64Q 3.9E−11G44R, CDR2 HzTFPI4F36 HC-FR2, A40T, G42E, G44R, Y59F, >3E−11 CDR2 A60P,K64Q HzTFPI4F36 HC-G42E, G42E, G44R, S49A, Y59F, >3E−11 G44R, S49A CDR2A60P, K64Q HzTFPI4F36 HC-G42E, G42E, G44R, A60P, K64Q 9.3E−11 G44R,A60P, K64Q HzTFPI4F36 HC-G44R, G44R, A60P, K64Q 3.0E−11 A60P, K64QThe mutations in both LC and HC as listed in tables 2 and 3 areconsistently numbered according to the Kabat numbering scheme as shownin FIG. 1.

The LC mutants listed in table 2 were expressed as LC mutants onlytogether with wild type HC HzTFPI4F36 CDRgrafted. The HC mutants listedin table 3 were expressed as HC mutants only together with wild type LCHzTFPI4F36 CDRgrafted. LC-HC combination mutants were also expressed bycombining different LC and HC mutants. Mutants are consistently namedafter the mutated chain, i.e. the final humanized mAb variant isexpressed with wild type HzTFPI4F36-CDRgrafted LC and the mutatedHzTFPI4F36 FR2, S49A, CDR2HC. Transient HEK293-6E expression wasperformed as described above.

The initial set of 9 point mutants (HzTFPI4F36 LC-S63T & HzTFPI4F36HC-Q3E; G44R; S49A; Y59F; A60P; K64Q; S77T; A93T) were based on aprimary set of back mutations highlighted in the humanization design.None of the point mutants rescued the affinity of the antibody, howevermutations in the second human heavy chain framework region (FR2, betweenCDR H1 and CDR H2) and in the C-terminal region of CDR H2 (omitted inthe minimal CDR grafting scheme) were highlighted as being important forTFPI binding. Affinity measurements by Biacore analyses were performedas described above.

The subsequent rounds of mutagenesis included a number of patch mutantsin which all residues in individual regions were mutated collectively.The mutant HzTFPI4F36 HC-Kabat CDR2, has 3 mutations Y59F, A60P, K64Q inthe C-terminal region of CDR H2, which corresponds to grafting CDR H2according to the Kabat definition and not according to the minimal CDRgrafting scheme used for the initial HzTFPI4F36-CDRgrafted variant. Thispatched mutant along with patch mutants in LC FR2 and HC FR2 improvedthe affinity of the humanized TFPI4F36 antibody significantly, butneither of the three patch mutants individually restored the highTFPI4F36 affinity.

The HC mutant with combined mutations in HC FR2, and CDR2 (A40T, G42E,G44R, S49A, Y59F, A60P, K64Q) did restore affinity completely. Thismutant introduced 7 additional murine residues into the antibodysequence. Combination of LC FR2 mutants (both FR2 mutations and Y36L)and HC FR2 and/or CDR2 mutants also resulted in high affinity mutants.However the combination of these LC/HC mutants consistently resulted inlower expression yields compared to the HC mutants alone. These resultsindicated that inclusion of the LC mutants had a negative impact on thestability of these antibody variants, hence suggesting that a delicateinteraction pattern between the humanized TFPI4F36 LC and HC exist.

In the last series of mutants, the 7 mutations in HC FR2, and CDR2(A40T, G42E, G44R, S49A, Y59F, A60P, K64Q) were dissected in order toeliminate potentially non-contributing back mutations. A series of 5mutants were generated to address this point.

In 3 mutants, back mutations were excluded in FR2:

-   -   HzTFPI4F36 HC-G42E, G44R, CDR2    -   HzTFPI4F36 HC—FR2, CDR2    -   HzTFPI4F36 HC-G42E, G44R, S49A CDR2

In 2 mutants additional mutations in CDR2 were also eliminated:

-   -   HzTFPI4F36 HC-G44R, A60P, K64Q    -   HzTFPI4F36 HC-G42E, G44R, A60P, K64Q

None of the mutants however, were on par with the combined HC FR2CDR2mutant. Either affinity or expression levels (or both) were impacted byany reduction in the HC FR2CDR2 mutant subset. The two mutantsHzTFPI4F36 HC G42E, G44R, Y59F, A60P, K64Q with 5 remaining backmutations and HzTFPI4F36 HC G42E, G44R, A60P, K64Q with 4 residual backmutations were picked for thorough comparison with HzTFPI4F36 HC—FR2,S49A, CDR2.

-   -   HzTFPI4F36 HC-G42E, G44R, A60P, K64Q and HzTFPI4F36 HC—FR2,        S49A, CDR2 expressed at comparable levels, while HzTFPI4F36        HC-G42E, G44R, CDR2 had a slightly lower expression level.        Accelerated biophysical stability studies did not show any        differences in stability in the three variants.    -   The affinities measured by Biacore are listed below.    -   In vivo efficacy measured in the dPT assay (as described below)        were comparable for all three variants.

TABLE 4 Kinetic parameters for the interaction between TFPI andhumanized TFPI4F36 variants. K_(D) Expression No of back Mutant (pM)yield (mg/L) mutations HzTFPI4F36 A40T, G42E, G44R, S49A, 26 54 7 Y59F,A60P, K64Q HzTFPI4F36 G42E, G44R, Y59F, 39 24 5 A60P, K64Q HzTFPI4F36G42E, G44R, A60P, K64Q 93 53 4

Based on the data described above, the original HC FR2, and CDR2 mutantwith 7 HC back mutations (A40T, G42E, G44R, S49A, Y59F, A60P, K64Q)tested superior to other variants; this variant is herein referred to asHzTFPI4F36 or as mAbTFPI2021.

It is likely that the CDR2 mutations Y59F, A60P, K64Q affect antibodyaffinity by directly interacting with antigen. Mutations A40T, G42E,G44R reside in a FR2 turn connecting CDRH1 and CDR H2, remote from theantigen binding face and could be poised for stabilizing LC-HCinteractions. The mutation S49A is buried in the middle of a highlyhydrophobic cluster of side chains which could explain why alanine ispreferred over serine at this position Interestingly therefore, the highaffinity of HzTFPI4F36 is obtained as a combination of mutations whichimprove the direct antigen interaction and mutations remote from theantigen binding region which stabilize the antibody.

In conclusion, HzTFPI4F36 has an affinity (K_(D)) of ˜25 μM and contains35 amino acid residues derived from the mouse antibody sequence,corresponding to 5.2% of the total number of residues in the antibody.

The amino acid sequences for the variable light (VL) region, variableheavy (VH) region, light chain and heavy chain of a selected humanizedconstruct, HzTFPI4F36 (mAbTFPI 2021), are shown in SEQ ID NOs: 15, 18,21 and 24, respectively.

In-Vitro Efficacy Assays

The anti-TFPI4F36 antibody is capable of neutralizing TFPI-mediatedinhibition of coagulation factor Xa (FXa) and the complex of tissuefactor (TF) and factor VIIa (FVIIa). The activities of murine andhumanized TFPI4F36 antibody variants were measured in a diluteprothrombin time (dPT) test. The dPT assay was used for measuring theprocoagulant activity of anti-TFPI antibodies. Increasing plasmaconcentrations of anti-TFPI antibody shortens the dPT clotting time.

Example 4 Purification, Crystallization and Structure of theFab-Fragment of MuTFPI4F36 (Fab) and the Second Kunitz Domain (K2) ofHuman Tissue Factor Pathway Inhibitor (TFPI)

A fragment of TFPI including its second Kunitz domain (K2) and aC-terminal His₆-tag (SEQ ID NO: 2) was co-crystallized with theMuTFPI4F36 Fab fragment (Fab). The structure of the complex was solvedby X-ray crystallography. The K2 binding epitope was found to becomposed of residues E10, E11, P13, R17, Y19, T21, Y23, Q28, Q31, E33,R34, F35, K36 and L50. The paratope in the Fab was found to compriseresidues E31, S32, D33, Y37, A96, T97, H98 and F99 of the MuTFPI4F36light chain (SEQ ID NO: 4) and residues N31, R53, S54, S56, Y57, Y59,F60, P61, D62, Q65, Y102, D103 and D106 of the MuTFPI4F36 heavy chain(SEQ ID NO: 8).

Materials and Methods Analytical Size Exclusion Chromatography.

Analytical size exclusion chromatography (SEC) was performed using aBiosep S-3000 (300×7.80 mm) column (Phenomenex) eluted with PBS-buffer(10 mM phosphate, 150 mM NaCl, 3 mM KCl, pH 7.5) at a flow rate of 0.8ml/min.

Preparation and Purification of the Fab/K2 Complex.

The Fab/K2 complex was prepared by mixing Fab (0.27 mg/ml in PBS buffer,pH 7.4) and K2 (0.29 mg/ml in PBS, pH 7.4) in a molar ratio of 1:1.5(5.4 mg Fab and 1.4 mg K2). The complex was concentrated on acentrifugal filter device (Amicon, 10 kD mw cut-off) to a concentrationof ˜6.7 mg/ml. To remove excess K2, the concentrated sample was appliedto a Superdex 75 (CV300) gel filtration column eluted with PBS-buffer,pH 7.4 at a flow rate of 1 ml/min. Fractions containing the Fab/K2complex were pooled and concentrated to a protein concentration of 9.2mg/ml. This solution was used for crystallization.

Crystallization of the Fab/K2 Complex.

The Fab/K2 complex was crystallized as rods by the hanging drop methodusing a precipitant solution containing 0.2 M tribasic potassium citrate(pH 8.0) and 20% w/v PEG 3,350.

Crystal Structure Determination.

The structure of the Fab/K2 complex was solved by the molecularreplacement method using PDB structures 1F8T and 1TFX as templates forthe Fab and K2 molecules, respectively.

Results

The complex between Fab and K2 was prepared by adding excess of K2 to asolution of Fab. FIG. 6 shows the complex formation monitored byanalytical size exclusion chromatography (SEC). This method separatesmolecules according to their molecular size with the larger specieseluting earlier than smaller. The peaks corresponding to K2 and Fab werewell separated due to the large difference in molecular weight (mw ˜8kDa and 48 kDa, respectively). Addition of K2 to the Fab solutionresulted in the expected minor shift in the peak position towardsshorter retention times. The complex was easily separated and obtainedin pure form by separating excess K2 using preparative SEC.

Conditions for crystallization of the Fab/K2 complex were screened usingseveral commercial crystallization screens. The hanging drop methodafforded rod-shaped crystals suitable for single crystal X-ray analysisand the structure was solved by the molecular replacement method usingstructures deposited in the PDB as templates. FIG. 7 shows the overallstructure of the Fab/K2 complex. Displayed are the light and heavychains, constituting the Fab molecule, and exhibiting the expectedimmunoglobulin β-sandwich fold characteristic for antibody molecules.Also shown are the CDR loops making contact with the antigen anddefining the specificity and affinity of the antibody.

The antigen, K2, exhibits the characteristic single anti-parallelβ-sheet (131 (120-N26) and β2 (Q31-Y37)) and the N-terminal α-helix (α1,L50-I56) that defines the Kunitz-fold (FIG. 8). Present is also theoptional 3₁₀-helix near the N-terminus (α0, D5-F8) followed by loop 1(L1, L9-Y19) leading to 131, which is connected to β2 via a short loop(Lβ, N27-K30). In the C-terminal segment, loop 2 (L2, G38-T49) connectsβ2 with al. Finally, the characteristic three disulfide bonds (C7-C57,C16-C40 and C33-C53) connect α0 with α1, L1 with L2 and β2 with α1,respectively.

Two structures of K2 have been deposited in the Worldwide ProteinDataBank (PDB). One structure, 1ADZ, is determined by NMR spectroscopyand represents the free solution structure, whereas the other, 1TFX, isdetermined by X-ray crystallography and represents K2 complexed withporcine trypsin. FIG. 9 shows the structural superposition of K2represented by 1ADZ, 1TFX and the K2 molecule in complex with Fab. Theback-bone traces appear very similar among all three structures,suggesting that the Kunitz-fold with its three stabilizing disulfidebonds is rather rigid.

Description of the MuTFPI4F36 K2 Binding Epitope

The binding epitope on the antigen K2, defined as residues in K2containing at least one side-chain heavy atom situated within a distanceof 4 Å or less from a heavy atom in Fab, comprises residues E10, E11,P13, R17, Y19, T21, Y23, Q28, Q31, E33, R34, F35, K36 and L50 (FIG. 10).The contact residues in K2 are located in L1 (E10, E11, P13, R17, Y19),in the β-sheet structure (T21, Y23, Q31, E33, R34, F35, K36) and theconnecting loop, Lβ (Q28) and, finally, a single one in α1 (L50). FIG.10 depicts the binding epitope mapped on to both the 3D-structure of K2and the primary amino acid sequence.

Description of the MuTFPI4F36 Paratope

The paratope in the MuTFPI4F36 Fab fragment was determined from the sameX-ray structure of the complex between the MuTFPI4F36 Fab and the TFPIK2 domain. The paratope was defined as those residues in the MuTFPI4F36Fab having a heavy atom within a distance of less than 4 Å from a heavyatom in the K2 domain. The contact residues in the light chain arelocated at residues E31, S32, D33, Y37, A96, T97, H98 and F99 of SEQ IDNO: 4. The contact residues in the heavy chain are located at residuesN31, R53, S54, S56, Y57, Y59, F60, P61, D62, Q65, Y102, D103 and D106 ofSEQ ID NO: 8. The location of the paratope is illustrated in FIG. 3.

Example 5 Structure of the K2/HzTFPI4F36 Fab Complex

Using methodology similar to that described for determination of thethree-dimensional structure of MuTFPI4F36 Fab bound to K2, the structureof the complex between the Fab fragment from the humanized antibody,HzTFPI4F36, and K2 was determined The Fab of HzTFPI4F36 was expectedlyfound to bind to the same region on K2 as the murine Fab from which itis derived. The overall similarity between the structures of the twocomplexes is evident in FIG. 11, where back bone ribbon traces areoverlaid for the K2/TFPI4F36 Fab and K2/HzTFPI4F36 Fab complexes. Theepitope (defined using a 4 Å cut-off) on K2 was, for HzTFPI4F36, foundto comprise residues E10, E11, D12, P13, R17, Y19, T21, Y23, F24, N26,Q28, Q31, C32, E33, R34, K36 and L50. In comparison with the structureof K2/MuTFPI4F36 Fab of murine origin, D12, F24, N26 and C32 are in thehumanized K2/HzTFPI4F36 complex within the 4 Å cut-off, whereas F35 isoutside. This reflects minor differences in side chain orientationswithin the binding interfaces of the K2/MuTFPI4F36 Fab and K2/HzTFPI4F36Fab complexes, in spite of the fact that the CDR regions in MuTFPI4F36and HzTFPI4F36 are identical.

Examples 6 to 8

The function of HzTFPI4F36 (mAbTFPI 2021) was compared to the functionof all (four) commercially available monoclonal antibodies, some ofwhich are said to bind to the K2 domain of TFPI; some of which have notbeen described with respect to binding.

Example 6 TFPI Neutralizing Assay: FXa Inhibition

Materials used were BSA buffer in assay (50 mM Hepes; 0.1 M NaCl, 5 mMCaCl₂, 0.1 mg/ml BSA, pH 7.4) and the reagents shown in table 5.

TABLE 5 Materials used Final concentration Company/ (dilution in ReagentReference Stock conc BSA buffer) Human FXa Enzyme Research 21.7M 5 nMLaboratory Human TFPI Reference: Freeze-dried in 6 nM Pedersen et al.,10 mM glycylglycine, 1990, J. Biol. 100 mM NaCl; Chem. 265, 165 mMmannitol p. 16786-16793 buffer pH 7.0. Reconstitute in water.mAbTFPI4F36 Current invention 5-150 nM mAb0281 Ab systems mAb4904 ADmAb2974 R&D systems mAb29741 R&D systems S2765 Chromogenix 20 mM 1 mM

Method:

Recombinant full length human TFPI (final concentration 6 nM)) was mixedin BSA buffer with increasing concentrations of the mAb of interest(final concentration: 5-150 nM) for 30 min. FXa was added and incubated30 min with the mixture for another 30 mM. Chromogenic substrate S2765was added and the absorption at 405 nm was measured for 15 min in aSpectramax. 100% activity represents the activity of FXa withoutaddition of TFPI.

TABLE 6 Neutralization of TFPI inhibition of FXa % neutralization ofTFPI Company mAb ID IC₅₀ nM at 150 nM Current invention HzTFPI4F36 12.6100% (mAbTFPI2021) AbNova mAb0281 nd <10% American Diagnotica mAb4904 nd<10% R&Dsystems mAb2974 29.4  90% R&Dsystems mAb29741 nd <10%

Conclusion:

At 150 nM, HzTFPI4F36 (mAbTFPI 2021) fully neutralized TFPI inhibitionof FXa. Almost no activity was detected for mAb0281, mAb4904 andmAb29741.

Example 7 TFPI Neutralizing Assay: FVIIa/TF/FXa Inhibition

Materials used were BSA buffer (50 mM Hepes; 0.1 M NaCl, 5 mM CaCl₂, 0.1mg/ml BSA, pH 7.4) EDTA: 50 mM and the reagents listed in table 7.

TABLE 7 Stock Final concentration Reagent Company/Reference Conc (dilutein BSA buffer) MAB2974 R&D systems 3330 nM Varying (5-150 nM)mAbTFPI4F36 Current invention 75300 nM NovoSeven Novo Nordisk 27 μM 1 pMvesicles HTI Phospholipids vesicles cat #PCPS- 2.0 mM 10M 02 #W1115-75%PC-25% PS S-2765 Chromogenix 35 mM 0.5 mM FX American Diagnostica inc.165 μM 160 nM Bovine factor X Product no 510 Lot No. 050920 dissolved50% glycerol/water TFPI Reference: Pedersen et al., 1990, J. Biol. 18.6μM 1 nM Chem. 265, p. 16786-16793 TF (Innovin) Dade Behring #2010-01-11#536975 vial 2.8 nM 1 pM diss. in 10 ml H2O (6 nM)

Method:

Add all the components in the final concentrations indicated in thetable. Add 25 μl FX, 25 μl TFPI mAb in varying concentrations, 25 μlhuman TFPI, 25 μl FVIIa-TF (innovin) in microtiter wells. Incubation for40 min at room temperature. Add 50 μl EDTA followed by 50 μl S-2765. Mixand read the plate for 15 min at 405 nm in Spectramax. 100% activity isthe activity of FVIIa/TF/FX obtained with no TFPI present.

TABLE 8 Neutralization of TFPI inhibition of FVIIa/TF/FX %neutralization of TFPI Company mAb ID IC₅₀ at 150 nM Current inventionHzTFPI4F36  3.8 nM 100% (mAbTFPI2021) AbNova mAb0281 nd Nd AmericanDiagnotica mAb4904 nd Nd R&Dsystems mAb2974 45.6 nM  53% R&DsystemsmAb29741 nd Nd

Conclusion:

At a mAb concentration of 150 nM TFPI is fully neutralised bymAbTFPI2021. mAb2974 also reaches saturation but does not fullyneutralize TFPI (53% neutralisation).

Example 8 Binding Interaction Analysis

Materials used were as listed in table 9.

TABLE 9 Reagent Company TFPI Freeze-dried in 10 mM glycylglycine, 100 mMNaCl; 165 mM mannitol buffer pH 7.0. Reconstitute in water. mAbTFPI2021Current invention mAb0281 Ab systems mAb4904 AD mAb2974 R&D systemsmAb29741 R&D systems All other reagents Biacore

Method:

Binding interaction analysis was obtained by Surface Plasmon Resonancein a Biacore T-100 instrument. Capture of the relevant monoclonalantibody at a fixed concentration was obtained by direct immobilizationto a CM5 chip of the mAb to a level of 500-1000 RU in 10 mM sodiumacetate pH 4.5-5.0. Four-fold dilutions of recombinant human full lengthTFPI or human TFPI short form (1-161 amino acid residues) from 200 nM to0.2 nM were tested for binding to the immobilized mAb. Running anddilution buffer: 10 mM HEPES, 150 mM, 0.005% p20, pH 7.4. Regenerationwas obtained by 10 mM Glycine, pH 1.7. Determination of kinetic andbinding constants (lc., k_(off), K_(D)) was obtained assuming a 1:1interaction of TFPI and the antibody of interest using the Biacore T100evaluation software. Results are shown in table 10. Competition of thedifferent mAbs for binding to TFPI when bound to mAbTFPI2021 (“mAb2021”,HzTFPI4F36) was obtained by immobilisation of mAbTFPI2021 to 5000 RU ata CM5 chip followed by binding of 50 nM TFPI followed by varyingconcentrations the mAbs (2974, 0281, 4904, 29741) to be tested forcompetition. Results are shown in table 11. Regeneration of the chip wasobtained by 10 mM Glycine, pH 1.7.

TABLE 10 Surface Plasmon Resonance (SPR) analysis. Binding to fulllength human TFPI. Kinetic and binding constants. Producer mAb ID ka(1/Ms) kd (1/s) K_(D) (M) K_(D) nM Current mAb2021 2.39E+06 3.58E−051.50E−11 0.015 invention AbNova mAb0281 3.99E+05 0.001436 3.60E−09 3.60American mAb4904 1.42E+05 00.1294 9.14E−09 9.14 Diagnotica R&DsystemsmAb2974 1.39E+06 0.001202 8.64E−10 0.864 R&Dsystems mAb29741 9.51E+050.003165 3.33E−09 3.33

TABLE 11 SPR analysis. Binding constant for binding to full length humanTFPI and TFPI₁₆₁(K1 and K2 domains). Competition with mABTFPI 2021.Competition K_(D) (M) K_(D) (M) with Producer mAb ID TFPI TFPI₁₆₁ mAB4F36 Current mAbTFPI2021 1.50E−11 4.55E−11 Yes invention AbNova mAb02813.60E−09 7.28E−09 No American mAb4904 9.14E−09 No binding No DiagnoticaR&Dsystems mAb2974 8.64E−10 3.12E−09 Yes R&Dsystems mAb29741 3.33E−09 Nobinding No

Conclusion

mAbTFPI2021 binds to TFPI with a higher affinity than any of the othermAbs tested (K_(D) 15 pM). Only mAb2974 competes for binding to samesite as mAb TFPI4F36.

Example 9 Neutralization of TFPI on Human Umbilical Vascular EndothelialCells (HUVECs)

Endothelial cells constitutively express TFPI in a form which isattached to the cell surface via a glycosylphosphatidylinositol (GPI)anchor. GPI-anchored TFPI specifically inhibits TF-mediated activitywhen TF is expressed on the same cell as TFPI. To demonstrate thatHzTFPI4F36 (mAbTFPI2021) neutralizes the inhibition by cell bound TFPImuch more efficiently than mAb 2974 we applied human umbilical vascularendothelial cells HUVECs; and in order to induce TF expression, thesecells were stimulated with TNFα (Sigma RBI) and IL113 (Roche) prior totesting of FVIIa/TF catalyzed activation of FX.

HUVEC cells were cultivated to confluence in 96 well plates in EBM-2medium (Clonetics) and stimulated with 20 ng/ml TNFα and 20 ng/ml IL1βfor 2 hours prior to testing. Testing was performed in 25 mM HEPES, 137mM NaCl, 3.5 mM KCl, 5 mM CaCl, 1 mg/ml BSA (0.1%) ph 7.4, and FXactivation was followed in the presence of antibody (0-20 nM) and withaddition of 50 μM FVIIa and 50 nM FX. Generation of FXa was measuredwith 0.6 mM of a chromogenic substrate, S-2765 (Chromogenix) andcalibrated towards a FXa standard curve.

FIG. 12 shows the results when the inhibition by cell bound TFPI wasabolished by 0-20 nM of HzTFPI4F36 or mAb 2974. TF/FVIIa-mediatedactivation of FX was stimulated by HzTFPI4F36 with a half maximal effectconcentration, (EC50˜nM) whereas hardly any stimulation of FXageneration was observed with the 2974 mAb at 20 nM.

Thus, this example illustrates that HzTFPI4F36, contrary to mAb 2974,efficiently neutralizes inhibition of TF/FVIIa-mediated FX activation bycell bound TFPI.

Example 10 Neutralization of TFPI Inhibition of TF/FVIIa Activity OmMDA-MB 231 Human Breast Carcinoma Cells

MDA-MB 231 cells constitutively express high levels of TF andinsignificant amounts of TFPI on the surface. Cell surface TF/FVIIamediated activation of FX can be inhibited by exogenous added TFPI. Todemonstrate that HzTFPI4F36 neutralizes this type of TFPI inhibitionmuch more efficiently than mAb 2974 we applied MDA-MB 231 cells andtested the ability of various concentrations of antibody to abolish theTFPI inhibition of FVIIa/TF catalyzed activation of FX.

MDA-MB 231 cells were cultivated to confluence in 96 well plates in DMEMGibco cat#31966-021 supplied with 10% FCS and 1% P/S. Testing wasperformed in 25 mM HEPES, 137 mM NaCl, 3.5 mM KCl, 5 mM CaCl, 1 mg/mlBSA (0.1%) ph 7.4, and FX activation was followed in the presence ofantibody (0-20 nM) and with addition of 2.5 nM full length humanrecombinant TFPI, 100 μM FVIIa and 50 nM FX. Generation of FXa wasmeasured with 0.6 mM of a chromogenic substrate, S-2765 (Chromogenix).The absorbance at 405 nm was measured continuously and the FXa activitywas determined by measuring the slope of the progress curve at 15 minafter initiation of the reaction.

FIG. 13 shows the results when the inhibition by TFPI was abolished by0-20 nM of HzTFPI4F36 or mAb 2974. TF/FVIIa-mediated activation of FXwas stimulated by HzTFPI4F36 with a half maximal effect concentration,(EC₅₀˜2 nM) whereas stimulation of FXa generation was obtained at asubstantially higher concentration of the 2974 mAb (EC₅₀>20 nM).

Example 11 Mapping the Binding Epitopes of the Anti-TFPI MonoclonalAntibodies, HzTFPI4F36 and mAb2974, Using ELISA

The binding epitope for HzTFPI4F36 on TFPI Kunitz-domain 2 (K2) has beenmapped by solving the crystal structure of the TFP1-K2/HzTFPI4F36complex. The effect of mutating single amino acid residues in TFPIwithin (E10, R17 and Y19) and outside (D5) the binding epitope forHzTFPI4F36 on the binding affinity to HzTFPI4F36 and mAb2974(R&Dsystems) was analyzed by ELISA. The TFPI variants were expressed inHEK293-F cells and the ELISAs were carried out using the conditionedmedium from the cell cultures.

The concentrations of TFPI-WT and TFPI mutants were estimated by anELISA, which bind TFPI K1 (MAb4903, American Diagnostica) and K3(MAb4F110, in-house) and hence is not affected by the mutations. Theeffect of the mutations on binding to HzTFPI4F36 was analyzed usingMAb4903 and HzTFPI4F36 in the ELISA. The effect on MAb2974 binding wasdetermined using an ELISA with MAb2974 and MAb4F110.

The effects of the mutations in TFPI-Kunitz 2 on binding to HzTFPI4F36and MAb2974 respectively, were calculated relative to TFPI-WT (100%binding) and illustrated in FIG. 14. The numbers have been corrected fordifferences in expression levels.

Conclusion:

Alanine mutation of the three amino acid residues within the bindingepitope for HzTFPI4F36 resulted in reduced binding to HzTFPI4F36,whereas alanine substitution of the residue located outside the epitope(TFP1-D5A) had no effect. Only one of the four alanine mutants,TFP1-Y19A, had reduced binding to MAb2974.

In conclusion, HzTFPI4F36 and MAb2974 have distinct but overlappingbinding epitopes located on TFPI-Kunitz 2.

Example 12 In Vivo Studies

Rabbits were made transiently haemophilic by intravenous administrationof 2000 RBU/kg of monoclonal anti-FVIII-antibodies. After 10 minutes,the rabbits received 12000 U/kg of anti-TFPI-antibody (4F36; 1.93mg/kg). Cuticle bleeding was induced 45 minutes afteranti-FVIII-antibody administration.

The 4F36 antibody caused a significant reduction in cuticle bleedingtime(FIG. 15). Administration of the 4F36 antibody led to no significantdrop in platelet number (FIG. 18).

A similar experiment was repeated in which three groups of eighttransiently haemophilic rabbits received either isotype control antibody(negative control group), 2 mg/kg anti-TFPI (mAb 4F36) or 9 mg/kgNovoSeven (positive control group) 5 minutes after cuticle bleeding wasinduced. Results are illustrated in FIG. 16: administration of mAB 4F36resulted in a considerable reduction in blood loss (approximately 85%)in all recipients, demonstrating that mAb4F36 can be used “on demand”.

Example 13 Estimation of Dose-Effect Relationship in Rabbit

The dose-effect relationship of the humanized mAb HzTFPI4F36 wasexamined in a rabbit haemophilia model. Rabbits were made transienthaemophilic by iv administration of a monoclonal anti-FVIII-antibody.After 10 minutes, the rabbits received 0.5, 1, 2 mg/kg HzTFPI4F36 or anisotype control antibody. After another 35 minutes cuticle bleeding wasinduced, followed by a 60 minutes observation period. HzTFPI4F36significantly and dose-dependently reduced bleeding time as well asblood loss when increasing the dose from 0.5 to 2 mg/kg (FIG. 17). Thus,a significant reduction of both bleeding time and blood loss wasachieved with 1 mg/kg HzTFPI4F36, corresponding to a plasmaconcentration of 18780 ng/ml HzTFPI4F36. Normalization of the bleedingwas achieved at 2 mg/kg, corresponding to a plasma concentration of30980 ng/ml HzTFPI4F36.

These data indicate that the ‘efficacious concentration’, e.g. theplasma concentration needed for normalization in the present model—ofHzTFPI4F36 is in the range of 18780 and 30980 ng/ml.

Example 14 PK/PD in Rabbits—‘Duration of Action’

A pharmacokinetic study of HzTFPI4F36 in rabbits dosed with 20 mg/kg wasperformed. At predetermined time-points during the study blood sampleswere drawn from the rabbits for pharmacokinetic profiling by an ELISAmeasuring free HzTFPI4F36 (shown in FIG. 3 below). Effect studies wereperformed at 4 days (96 hours), 7 days (168 hours) and 10 days (240hours) after administration, using the cuticle bleeding model intransient haemophilic rabbits, the effect time points are indicated inFIG. 19.

The pharmacokinetic profile is biphasic indicative of target mediatedclearance. Thus, above the bend of the curve excess free mAb is present(mAB_(free)>TFPI_(total)), below the bend: mAb_(free)<TFPI_(total). Ingood accordance with the pharmacokinetic profile, both bleeding time andblood loss was significantly reduced both at 4 and 7 days afteradministration of 20 mg/kg HzTFPI4F36 intravenously, whereas nosignificant effect was observed after 10 days (FIG. 20).

These data confirm that the efficacious plasma concentration ofHzTFPI4F36 in a cuticle bleeding model in haemophilic rabbits is between18780 and 30980 ng/ml which is close to the TFPI saturation limit (curvebend). Accordingly, a single iv. dose of 20 mg/kg HzTFPI4F36 reducedcuticle bleeding for at least 7 days, which corresponded to the periodof time the plasma concentration was above the ‘efficaciousconcentration’

Example 15 Pharmacokinetic Model Based on Monkey PK Data

A pharmacokinetic evaluation was made based on a pharmacokinetic studyin monkeys, where both single and multiple doses were administered (FIG.21). Dose levels ranged from 2 to 200 mg/kg.

The PK profile in monkey (20 mgs/kg, upper panel) is similar to rabbitindicating the presence of similar distribution of soluble andendothelium bound TFPI. Thus, these data indicate that rabbit effectdata may be employed to predict the effect range in monkey. Furthermore,the affinity of HzTFPI4F36 for human, monkey and rabbit TFPI are similar(same epitope) and similar TFPI tissue distribution in the three speciesallows for dose predictions in monkey and man.

Example 16 Simulations

Based on the model presented above, it was possible to make a series ofsimulations. The main objective of the simulations was to describe theoptimal dosing regimen in a multiple dose setting. The target (TFPI)concentration was not known, but the rabbit effect data above allows forthe assumption that if the target is near saturation at a level of 30000ng/ml then full effect in a bleeding model is obtained.Therefore, themain objective of the simulations was to evaluate which dose levels overa period of time would lead to full saturation. FIG. 22 displays asimulation of 1 mg/kg administered subcutaneously. FIG. 23 shows asimulation of 15 mg/kg HzTFPI4F36 (mAbTFPI 2021) administered IV everythird week. FIG. 24 shows a simulation of 20 mg/kg HzTFPI4F36 (mAbTFPI2021) administered IV every second week.

In summary, based on the above simulations the following dose regimenprediction can be made for human beings:

TABLE 12 Dose regimen Type of dose Dose Dose regimen S.c. adm 1 mg/kgEvery 2^(nd) day I.v. adm 10-20 mg/kg Every 2^(nd)-4^(th) week

1. A monoclonal antibody that is capable of specifically binding the K2domain of TFPI, wherein said antibody is capable of specifically bindingan epitope comprising residue R17 of SEQ ID NO:
 2. 2. The monoclonalantibody of claim 1, wherein the epitope of said antibody comprises oneor more residues selected from the group consisting of E10, E11, D12,P13, R17, Y19, T21, Y23, F24, N26, Q28, Q31, C32, E33, R34, F35, K36,and L50 of SEQ ID NO:
 2. 3. The monoclonal antibody of claim 1, whereina light chain of said antibody comprises amino acid residues: E, in theposition corresponding to position 31, S, in the position correspondingto position 32, D, in the position corresponding to position 33, Y, inthe position corresponding to position 37, A, in the positioncorresponding to position 96, T, in the position corresponding toposition 97, and F, in the position corresponding to position 99 of SEQID NO: 15; and wherein a heavy chain of said antibody comprises aminoacid residues: N, in the position corresponding to position 31, R, inthe position corresponding to position 53, S, in the positioncorresponding to position 54, Y, in the position corresponding toposition 57, Y, in the position corresponding to position 59, F, in theposition corresponding to position 60, P, in the position correspondingto position 61, D, in the position corresponding to position 62, Q, inthe position corresponding to position 65, Y, in the positioncorresponding to position 102, D, in the position corresponding toposition 103, and D, in the position corresponding to position 106 ofSEQ ID NO
 18. 4. The monoclonal antibody of claim 3, wherein said heavychain further comprises an S in the position corresponding to position52 of SEQ ID NO:
 18. 5. The monoclonal antibody of claim 4, wherein saidlight chain further comprises an H in the position corresponding toposition 98 of SEQ ID NO: 15 and said heavy chain further comprises an Sin the position corresponding to position 56 of SEQ ID NO:
 18. 6. Themonoclonal antibody of claim 1, wherein a heavy chain of said antibodycomprises: a CDR1 sequence of amino acids 31 to 35 (NYAMS) of SEQ ID NO:18, wherein one of these amino acids may be substituted by a differentamino acid; and/or a CDR2 sequence of amino acids 50 to 66(TISRSGSYSYFPDSVQG) of SEQ ID NO: 18, wherein one, two or three of theseamino acids may be substituted by a different amino acid; and/or a CDR3sequence of amino acids 99 to 110 (LGGYDEGDAMDS) of SEQ ID NO: 18,wherein one, two or three of these amino acids may be substituted by adifferent amino acid.
 7. The monoclonal antibody of claim 1, wherein thelight chain of said antibody comprises: a CDR1 sequence of amino acids24 to 39 (KSSQSLLESDGKTYLN) of SEQ ID NO: 15, wherein one, two or threeof these amino acids may be substituted with a different amino acid;and/or a CDR2 sequence of amino acids 55 to 61 (LVSILDS) of SEQ ID NO:15, wherein one or two of these amino acids may be substituted with adifferent amino acid; and/or a CDR3 sequence of amino acids 94 to 102(LQATHFPQT) of SEQ ID NO: 15, wherein one or two of these amino acidsmay be substituted with a different amino acid.
 8. The monoclonalantibody according to claim 1, wherein a light chain of said antibodycomprises SEQ ID NO: 21 and a heavy chain of said antibody comprises SEQID NO:
 24. 9. The monoclonal antibody according to claim 1, wherein themonoclonal antibody is capable of binding the K2 domain of TFPI with ahigher affinity than mAb2974.
 10. A monoclonal antibody according toclaim 1 that is capable of neutralising the TFPI inhibition ofmembrane-bound FVIIa/TF/FXa by at least 55% as measured in anFVIIa/TF/FXa inhibitor assay, when TFPI is saturated with said antibody.11. The monoclonal antibody according to claim 1, wherein the monoclonalantibody is a humanized antibody.
 12. The monoclonal antibody accordingto claim 1, wherein the monoclonal antibody is a human antibody.
 13. Themonoclonal antibody according to claim 1, wherein the monoclonalantibody is a chimeric antibody.
 14. The monoclonal antibody accordingto claim 1, wherein the isotype of said antibody is IgG.
 15. Themonoclonal antibody according to claim 1, wherein said isotype isselected from the group consisting of IgG1, IgG2, and IgG4.
 16. Themonoclonal antibody of claim 1, wherein the monoclonal antibody isselected from the group consisting of a Fab fragment, a F(ab′)₂fragment, a Fab′ fragment, a Fd fragment, a Fv fragment or a dAbfragment.
 17. The monoclonal antibody of claim 1, which is a deletionvariant or an insertion variant.
 18. A eukaryotic cell which expressesthe monoclonal antibody, or a fragment thereof, according to claim 1.19. A pharmaceutical composition comprising an antibody according toclaim 1 and a pharmaceutically acceptable carrier or diluent.
 20. Amethod of treating a subject with a coagulopathy, comprisingadministering to said subject the monoclonal antibody according to claim1.